Particles Comprising a Shell with RNA

ABSTRACT

The present invention relates to RNA decorated particles such as RNA decorated lipid particles, preferably to RNA decorated liposomes. Further, the present invention relates to a pharmaceutical composition comprising RNA decorated particles such as RNA decorated lipid particles, preferably RNA decorated liposomes. Said pharmaceutical composition is useful for inducing an immune response. It is also useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen. Furthermore, the present invention relates to a method for producing the RNA decorated particles such as RNA decorated lipid particles, preferably RNA decorated liposomes.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to RNA decorated particles such as RNAdecorated lipid particles, preferably to RNA decorated liposomes.Further, the present invention relates to a pharmaceutical compositioncomprising RNA decorated particles such as RNA decorated lipidparticles, preferably RNA decorated liposomes. The pharmaceuticalcomposition is useful for inducing or enhancing an immune response. Itis also useful in a prophylactic and/or therapeutic treatment of adisease involving an antigen. Furthermore, the present invention relatesto a method for producing the RNA decorated particles such as RNAdecorated lipid particles, preferably RNA decorated liposomes.

BACKGROUND OF THE INVENTION

Hydrophilic molecules such as nucleic acids or water-soluble drugs areoften carried by lipid vesicles providing a protective environment sothat said molecules can cross cell membranes and enter target cells.Lipid vesicles are substantially spherical structures made of materialshaving a high amphiphilic lipid content. Lipid vesicles are usuallycalled liposomes, if the lipid molecules are orientated in a lipidbilayer around an aqueous cave. Hydrophilic molecules but alsohydrophobic molecules as well as amphiphilic molecules can be carried byliposomes. In particular, hydrophilic molecules can be carried byliposomes being comprised in the aqueous internal space of theliposomes, hydrophobic molecules can be carried by liposomes beingcomprised in the lipid bilayer of the liposomes and amphiphilicmolecules can be carried by liposomes being comprised at the interfacebetween lipid bilayer and aqueous internal space of the liposomes.Liposomes can be distinguished by their form and size and can beclassified, for example, in multilamellar vesicles (MLV), largeunilamellar vesicles (LUV), small unilamellar vesicles (SUV) or in otherforms.

In the last decades, a wide range of liposome formulations have beeninvestigated for use in medical applications, cosmetics or foodindustry. The first most prominent liposome-based products are thecancer drugs Doxil (Sequus) and DaunoXome (Gilead, Nexstar), which havebeen approved by the US Food and Drug Administration (FDA) in the 1990s(Wagner, A., Vorauer-Uhl, K., (2011), Journal of Drug Delivery,2011:591325). Recent investigations resulted in the generation of newclasses of liposomes such as dendrosomes (Sarbolouki, M. N.,Sadeghizadeh, M., Yaghoobi, M. M., Karami, A., Lohrasbi, T. (2000),Journal of Chemical Technology and Biotechnology, 75, 919-922) orcationic liposomes (Audouy, S., Hoekstra, D. (2001), Molecular MembraneBiology, 18, 129-143). Cationic liposomes are structures that are madeof positively charged lipids and are increasingly being researched foruse in gene therapy due to their favourable interactions with negativelycharged DNA and cell membranes. Recently, cationic liposomes have beenprovided not only for carrying DNA molecules but also for carrying RNAmolecules or other therapeutically active compounds.

Disadvantages of current liposomes are that they need to be tailored fora given type of compound. For example, lipophilic, hydrophilic orpolymeric compounds need different lipidic carriers to obtain suitablepayload and targeting efficacy. One problem with water soluble compoundsis the susceptibility to leakage, for example on binding to proteins,peptides, polynucleic acids or polymers in general. Thus, there is aneed of improved formulations of particles for the delivery oftherapeutically active compounds.

As mentioned above, lipid particles, such as liposomes, have usuallytherapeutic active compounds encapsulated in their interior. The presentinventors surprisingly found that with particles having water-solublecompounds encapsulated in their lipid vesicular core RNA can be boundthereon, maintaining the vesicular organization, and maintaining,partially or completely the encapsulated compound. The RNA decorationdoes not lead to loss of the encapsulated therapeutically activecompound. It is known that RNA molecules are easily degraded in bodyfluids after systemic administration by ribonucleases. The presentinventors surprisingly found that the RNA on the RNA decorated particlesis stable and does not form aggregates.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a particlecomprising:

-   (i) a vesicular core,-   (ii) at least one therapeutically effective compound encapsulated    within the vesicular core, and-   (iii) RNA forming a hydrophilic shell on at least a portion of the    vesicular core.

In one embodiment, the particle has a net negative charge, a netpositive charge or is electroneutral.

In one embodiment, the RNA is pharmaceutically active or encodes atleast one pharmaceutically active peptide or protein. In one embodiment,the RNA encodes at least one antigen.

In one preferred embodiment, the antigen is a disease-associated antigenor elicits an immune response against a disease-associated antigen orcells expressing a disease-associated antigen.

In one embodiment, the RNA is exposed to surrounding medium.

In one embodiment, the RNA covers the entire surface of the vesicularcore or a portion thereof.

In one embodiment, the therapeutically effective compound is awater-soluble compound.

In one embodiment, the therapeutically effective compound is a smallmolecule compound.

In one embodiment, the therapeutically effective compound is useful inimmunotherapy.

In one embodiment, the therapeutically effective compound is an agentstimulating γδ T cells, preferably Vγ9Vδ2 T cells.

In one preferred embodiment, the agent stimulating γδ T cells is abisphosphonate. In one more preferred embodiment, the agent stimulatingγδ T cells is a nitrogen-containing bisphosphonate(aminobisphosphonate). In one even more preferred embodiment, the agentstimulating γδ T cells is selected from the group consisting ofzoledronic acid, clodronic acid, ibandronic acid, pamidronic acid,risedronic acid, minodronic acid, olpadronic acid, alendronic acid,incadronic acid and salts thereof.

In one embodiment, the vesicular core is positively charged.

In one embodiment, the vesicular core is a polymer vesicular core, aprotein vesicular core or a lipid vesicular core, preferably a lipidvesicular core.

In one particularly preferred embodiment, the invention relates toparticle comprising:

-   (i) a positively charged lipid vesicular core,-   (ii) at least one therapeutically effective compound encapsulated    within the vesicular core, and-   (iii) RNA forming a hydrophilic shell on at least a portion of the    vesicular core.

In one embodiment, the lipid vesicular core comprises a lipid bilayer.

In one embodiment, the lipid vesicular core comprises a liposome.

In one embodiment, the lipid vesicular core comprises at least onecationic lipid.

In one preferred embodiment, the lipid vesicular core comprises aliposome comprising at least one cationic lipid.

In one embodiment, the positive charges are contributed by the at leastone cationic lipid and the negative charges are contributed by the RNA.

In one embodiment, the lipid vesicular core comprises at least onehelper lipid.

In one preferred embodiment, the helper lipid is a neutral lipid ornegatively charged lipid.

In one preferred embodiment, the at least one cationic lipid comprises1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In one more preferredembodiment, the at least one cationic lipid comprises DMEPC and/orDOTMA.

In one preferred embodiment, the at least one helper lipid comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin(POPC) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In onemore preferred embodiment, the at least one helper lipid comprises DSPC,DOPE, and/or Chol.

In one even more preferred embodiment, the at least one cationic lipidcomprises DMEPC and the at least one helper lipid comprises DSPC andDOPE, or the at least one cationic lipid comprises DOTMA and the atleast one helper lipid comprises Chol.

In one embodiment the particle has an average diameter in the range offrom about 50 nm to about 1000 nm. In one embodiment the particle has anaverage diameter in the range of from about 300 nm to about 600 nm. Inone embodiment the particle has an average diameter of about 200 nm orless. Particles having an average diameter in the range of from about300 nm to about 600 nm are preferably useful for targeting antigenpresenting cells, preferably antigen presenting cells in the spleen,preferably professional antigen presenting cells such as dendriticcells. Particles having an average diameter of about 200 nm or less arepreferably useful for targeting tumor cells.

In one preferred embodiment, the particle has an average diameter

-   (i) in the range of from about 50 nm to about 400 nm, preferably    from about 50 nm to 200 nm, or-   (ii) in the range of from about 200 nm to about 1000 nm, preferably    from about 200 nm to about 800 nm, more preferably from about 300 nm    to about 600 nm.

In one embodiment, the lipid vesicular core having the therapeuticallyeffective compound encapsulated therein is obtainable by reverse phaseevaporation technique or ethanol injection technique.

In one embodiment, the particle is obtainable by addition of the RNA toa lipid vesicular core having the therapeutically effective compoundencapsulated therein.

In one embodiment, the particle is obtainable by a process comprising astep of extruding and/or a step of lyophilizing the particle.

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising particles according to the first aspect.

In one embodiment, after systemic administration of the particles, atleast a portion of the RNA and at least a portion of the therapeuticallyeffective compound are delivered to a target cell, preferably to thesame target cell. In one embodiment, the target cell is a spleen cell,preferably an antigen presenting cell, more preferably a professionalantigen presenting cell, more preferably a dendritic cell. Thus,particles of the invention may be used for delivering RNA and atherapeutically effective compound to such target cell.

In one embodiment, after systemic administration of the particles, RNAaccumulation and/or RNA expression in the spleen occurs. Thus, particlesof the invention may be used for expressing RNA in the spleen.

In one embodiment, after systemic administration of the particles, no oressentially no RNA accumulation and/or RNA expression in the lung and/orliver occurs.

In one embodiment, after systemic administration of the particles, RNAaccumulation and/or RNA expression in the spleen is at least 5-fold theamount of RNA accumulation and/or RNA expression in the lung.

In one embodiment, after systemic administration of the particles, RNAaccumulation and/or RNA expression in antigen presenting cells,preferably professional antigen presenting cells, in the spleen occurs.Thus, particles of the invention may be used for expressing RNA in suchantigen presenting cells.

In one preferred embodiment, the antigen presenting cells are dendriticcells and/or macrophages.

In one embodiment, systemic administration is by parenteraladministration, preferably by intravenous administration, subcutaneousadministration, intradermal administration or intraarterialadministration.

In one embodiment, the pharmaceutical composition further comprises oneor more pharmaceutically acceptable carriers, diluents, and/orexcipients.

In one embodiment, the pharmaceutical composition further comprises atleast one adjuvant.

In one embodiment, the pharmaceutical composition is formulated forsystemic administration.

In a third aspect, the present invention relates to the pharmaceuticalcomposition according to the second aspect for inducing or enhancing animmune response, preferably an immune response against cancer.

In a fourth aspect, the present invention relates to the pharmaceuticalcomposition according to the second aspect, for use in a prophylacticand/or therapeutic treatment of a disease involving an antigen,preferably a cancer disease.

In a fifth aspect, the present invention relates to a method fordelivering an antigen to antigen presenting cells, preferablyprofessional antigen presenting cells, in the spleen or expressing anantigen in antigen presenting cells, preferably professional antigenpresenting cells, in the spleen

comprising administering to a subject a pharmaceutical compositionaccording to the second aspect. In this aspect, the antigen or a portionthereof is preferably encoded by the RNA forming a hydrophilic shell onat least a portion of the vesicular core.

In one preferred embodiment, the antigen presenting cells are dendriticcells and/or macrophages.

In a sixth aspect, the present invention relates to a method forinducing or enhancing an immune response, preferably an immune responseagainst cancer, in a subject

comprising administering to the subject a pharmaceutical compositionaccording to the second aspect.

In a seventh aspect, the present invention relates to a method forstimulating, priming and/or expanding T cells in a subject

comprising administering to the subject a pharmaceutical compositionaccording to the second aspect.

In an eighth aspect, the present invention relates to a method oftreating or preventing a disease involving an antigen, preferably acancer disease, in a subject

comprising administering to the subject a pharmaceutical compositionaccording to the second aspect. In this aspect, the antigen or a portionthereof is preferably encoded by the RNA forming a hydrophilic shell onat least a portion of the vesicular core.

In a ninth aspect, the present invention relates to a method ofproducing a particle according to the first aspect comprising thefollowing steps of:

-   (i) providing a vesicular core having at least one therapeutically    effective compound encapsulated therein, and-   (ii) adding RNA to the vesicular core, wherein the RNA forms a    hydrophilic shell on at least a portion of the vesicular core,    thereby forming the particle.

Embodiments of the vesicular core, the therapeutically effectivecompound, the RNA and/or the particle produced are as described above.

In one embodiment, the vesicular core is a lipid vesicular core,preferably a positively charged lipid vesicular core.

In one preferred embodiment, the lipid vesicular core to which the RNAis added comprises a liposome comprising at least one cationic lipid.

In one preferred embodiment, the amount of RNA and the amount ofcationic lipids in the liposome is selected such that the net chargeformed by the positive charges derived from the cationic lipids and thenegative charges derived from the RNA is negative, positive, or zero.

In one even more preferred embodiment, the number of positive chargesderived from the cationic lipids divided by the number of negativecharges derived from the RNA is between 0.025 and 4, preferably is0.025, 0.125, 0.250, 0.375, 0.500, 0.625, 0.750, 0.875, 1, 2, 3, or 4.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Binding of RNA as a function of total cationic lipid (DOTMA) inthe liposomes.

Zoledronic acid (ZA) encapsulating liposomes (ZA liposomes) withdifferent compositions and molar fractions of the cationic lipid DOTMAwere prepared and the binding of RNA to these liposomes wasinvestigated. The liposome composition was as follows: DOTMA/CHOL/POPC10/50/40, DOTMA/CHOL/POPC 20/50/30, DOTMA/CHOL/POPC 30/50/20,DOTMA/CHOL/POPC 40/50/10, and DOTMA/CHOL/POPC 50/50/0 molar ratio,respectively. Thus, the liposomes were composed of 10%, 20%, 30%, 40%,or 50% DOTMA. Binding was investigated by adding an excess of RNA to thezoledronic acid (ZA) encapsulating liposomes (ZA liposomes) andquantifying the RNA by capillary electrophoresis (Bioanalyzer). TheDOTMA/RNA charge ratios were as follows: DOTMA/RNA (mole/base)=0.025,0.125, 0.25, 0.375, 0.50, 0.625, 0.75, 0.875, 1.00. When cationicliposomes were present, the measured amount of RNA decreased. Themissing RNA was taken as liposome bound RNA. As can be seen, the amountof bound RNA was directly proportional to the amount of DOTMA present ina one-to-one stoichiometry with respect to the charge. This means, forall tested liposomes, that the amount of bound RNA was directlycorrelated with the amount of DOTMA in the membrane. As the molarfraction of DOTMA changed (from 10% to 50%), also the amount of boundRNA per liposome and the surface coverage of the liposomes with RNAchanged. Thus, in the given experiment, RNA covered liposomes, where thesurface coverage with RNA changed by a factor of five, could beassembled in a controlled way.

FIG. 2: Particle sizes and polydispersity indices (PI) of cationicZA-liposomes after addition of RNA at different charge ratios: cationiclipid (DOTMA)/RNA.

The particle sizes and the polydispersity indices (PIs) of different RNAdecorated ZA-liposomes (ZARNAsomes) (i.e. RNA decorated zoledronic acid(ZA) encapsulating liposomes) and undecorated ZA-liposomes (i.e.zoledronic acid (ZA) encapsulating liposomes) were compared. Theliposomes had the following composition: DOTMA/CHOL/POPC in a 30/50/20molar ratio. An excess of negative charges and an excess of positivecharges was investigated. DOTMA to RNA in a 1/4, 1/2, 1/1, 2/1, and 4/1positive charge/negative charge-ratio (+/−) were tested. The sizes,indicated in Z-averages (Z_(ave)), and the polydispersity indices (PIs)of the particles were measured by Photon Correlation Spectroscopy. Ascan be seen, in case of an excess of negative charges (charge reversalwith respect to the precursor cationic liposomes), liposome formulationswith discrete particle sizes were obtained, and no aggregation wasobserved. Only moderate changes of the particle size with respect to theprecursor liposomes occurred, that may be in line with the boundmolecular layer on the liposome surface.

FIG. 3: Expression of luciferase (Luc) in vitro transcribed (IVT) RNA indendritic cells after incubation with liposome formulations.

The luciferase expression was evaluated via luminescence indicating themetabolic rate of luciferin as substrate for luciferase in counts perseconds (cps). In total, four donors were tested separately. The meanvalue is shown including the standard deviation (SD). Only luciferase(Luc) RNA decorated zoledronic acid (ZA) containing liposomes(ZARNAsomes) were stable and resulted in luciferase expression, incontrast to naked RNA or zoledronic acid (ZA) containing liposomeswithout RNA (ZA-L).

FIG. 4: Relative expression of maturation markers in dendritic cellsafter incubation with liposome formulations.

Here, the relative expression of CD83 (A), CD86 (B) and HLA-DR (C) isshown. Expression data were normalized to no stimulation control.ZARNAsomes resulted in a distinct higher expression of all markers.Regarding CD86 and HLA-DR, the expression was even comparable with thepositive control (pos. ctrl.). Mean values of two donors are shown.

FIG. 5: Functionality of encapsulated zoledronic acid (ZA) afterco-cultivation of immature dendritic cells (iDCs) incubated withliposome formulations and peripheral blood lymphocytes (PBLs).

(A) The frequency of Vγ9Vδ2 T cells regarding all lymphocytes is shown.A mean value of two donors is shown. (B) The expansion rate of Vγ9Vδ2 Tcells is shown. Total cell numbers after 7-day cultivation have beendivided by ex vivo amounts. Also the mean value of two donors is shown.Vγ9Vδ2 T cell frequency and expansion rate was increased in the presenceof ZARNAsomes.

FIG. 6: Application of ZARNAsomes resulted in luciferase expression inthe spleen.

Comparison of luciferase (Luc) RNA decorated zoledronic acid (ZA)encapsulating liposomes (ZARNAsomes) with luciferase (Luc) RNA decoratedbuffer vehicle encapsulating liposomes (EL+Luc-RNA) shows thatzoledronic acid (ZA) encapsulation does not negatively influence RNAuptake and translation. The liposomes had the following composition:DOTMA/Chol. (A) Bioluminescence imaging of mice 6 hours after i.v.injection of luciferase (Luc) RNA decorated zoledronic acid (ZA)encapsulating liposomes (Luc-RNA, 20 μg) (ZARNAsomes) or luciferase(Luc) RNA decorated buffer vehicle encapsulating liposomes (Luc-RNA, 20μg) (EL+Luc-RNA). (B) Quantification of in vivo spleen bioluminescencesignal (p/s, photons per second); *p<0.05 (t-test).

FIG. 7: Application of ZARNAsomes resulted in upregulation of CD40 andCD86 expression on splenic dentritic cells (DCs) and macrophage (mΦ)cell population.

Independent of which antigen the decorated RNA codes for, macrophage andDC maturation was induced in the presence of ZARNAsomes or buffervehicle encapsulating liposomes decorated with RNA (EL+respective RNA).In contrast thereto, undecorated ZA encapsulating liposomes (ZA-L),buffer vehicle encapsulating liposomes (EL) and free RNA did not lead tomacrophage and DC maturation. FACS analysis results of (A) DC-populationand (B) macrophage-population of splenocytes 24 hours after i.v.injection of luciferase RNA or influenza hemagglutinin (infHA) RNA (20μg) decorated ZA encapsulating liposomes (ZARNAsome Luc-RNA, ZARNAsomeinfHA-RNA), luciferase RNA or influenzaHA RNA (20 μg) decorated buffervehicle encapsulating liposomes (EL+Luc-RNA, EL+infHA-RNA), buffervehicle encapsulating liposomes (EL), and free luciferase RNA orinfluenzaHA RNA (free Luc-RNA or free infHA-RNA) are shown. Meanfluorescence intensities (MFI) of n=1-3 animals are presented. infHA-RNAor InfluenzaHA-RNA=influenza hemagglutinin A RNA.

FIG. 8: Application of carboxyfluorescin (CF) -filled liposomesdecorated with Luc-RNA (CF-filled ZARNAsome) resulted in transfection ofsplenic cell populations, whereby dendritic cells (DCs) and macrophages(m4)) were the main targets.

FACS analysis of splenocytes 1 hour after i.v. injection of CF-filledZARNAsomes complexed with Luc-RNA (20 μg) (CF filled ZARNAsome) showed aCF-signal increase, preferably in dendritic cells and macrophages.Displayed is the frequency of CF positive cells in % of parentpopulation of n=1-3 animals.

FIG. 9: Zoledronic acid resulted in an accumulation ofisopentenylpyrophosphate (IPP) in the spleen.

Application of zoledronic acid (ZA) encapsulating liposomes (ZA-L) andluciferase (Luc) RNA decorated zoledronic acid (ZA) encapsulatingliposomes (ZARNAsome Luc-RNA) resulted in an accumulation ofIsopentenylpyrophosphate (IPP) in splenocytes. In contrast thereto,application of free luciferase (Luc) RNA (free RNA), buffer vehicleencapsulating liposomes decorated with luciferase (Luc) RNA (EL+Luc RNA)and buffer vehicle encapsulating liposomes (EL) did not increase IPPvalues. Bars represent mean IPP values of 3 animals 24 h after i.v.administration, *p<0.05; **p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise. For example, if in a preferredembodiment the particle of the present invention comprises awater-soluble therapeutically effective compound and if in anotherpreferred embodiment the particle of the present invention comprises RNAencoding at least one antigen, it is a contemplated preferred embodimentthat the particle of the present invention comprises a water-solubletherapeutically effective compound and RNA encoding at least oneantigen.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps. The terms “a” and “an” and “the”and similar reference used in the context of describing the invention(especially in the context of the claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. Recitation of ranges of values hereinis merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”),provided herein is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

In the following, definitions will be provided which apply to allaspects of the present invention.

In the context of the present invention, the term “particle” relates toa structured entity formed by molecules or a molecule complex. In oneembodiment, the structured entity formed by molecules or a moleculecomplex comprises a positively charged lipid vesicular core, atherapeutically effective compound encapsulated within the vesicularcore, and RNA forming a hydrophilic shell on at least a portion of thevesicular core. The term “particle” in particular relates to a micro- ornano-sized spherical structure.

In one embodiment, the particles of the present invention have anaverage diameter in the range of from about 50 nm to about 1000 nm, e.g.from about 100 nm to about 900 nm, from about 200 nm to about 800 nm,from about 200 to about 700 nm, from about 300 to about 600 nm, fromabout 300 nm to about 500 nm, or from about 300 nm to about 400 nm.

In one embodiment, the particles of the present invention have anaverage diameter of at least about 50 nm, at least about 60 nm, at leastabout 70 nm, at least about 80 nm, at least about 90 nm, at least about100 nm, at least about 150 nm, at least about 200 nm, at least about 250nm, at least about 300 nm, at least about 400 nm, at least about 500 nm,at least about 600 nm, at least about 700 nm, at least about 800 nm, atleast about 900 nm, and/or the particles of the present invention havean average diameter of no more than about 1000 nm, no more than about900 nm, no more than about 800 nm, no more than about 700 nm, no morethan about 600 nm, no more than about 500 nm, no more than about 400 nm,no more than about 300 nm, no more than about 250 nm, no more than about200 nm, no more than about 150 nm, no more than about 100 nm, no morethan about 90 nm, no more than about 80 nm, no more than about 70 nm, nomore than about 60 nm.

In one preferred embodiment, the particles of the present invention havean average diameter (i) in the range of from about 50 nm to about 400nm, preferably from about 50 nm to about 200 nm, or (ii) in the range offrom about 200 nm to about 1000 nm, preferably from about 200 nm toabout 800 nm, more preferably from about 300 nm to about 600 nm. The useof particles having diameters≦about 200 nm is preferred for targetingtumor cells. In addition, the use of particles having diameters betweenabout 300 nm and about 600 nm is preferred for targeting antigenpresenting cells such as dendritic cells or macrophages.

In one embodiment, the particles of the present invention are comprisedin a formulation such a liquid formulation. Thus, the present inventionmay refer to a formulation such as a liquid formulation comprising theparticles of the present invention.

The term “vesicular core” refers to a vesicle structure capable ofencapsulating a therapeutically effective compound and capable ofproviding a binding surface on its outside for RNA. In other words, theoutside of the vesicular core is structured such that it can be coveredby RNA and the inside of the vesicular core is structured such that itfaces a lumen, in which a therapeutically effective compound can beencapsulated. The vesicular core may be a structure comprising orconsisting of and preferably formed by polymers, proteins and/or lipids.

The term “lipid vesicular core” refers to a lipid vesicle structurecapable of encapsulating a therapeutically effective compound andcapable of providing a binding surface on its outside for RNA. In otherwords, the outside of the lipid vesicular core is structured such thatit can be covered by RNA and the inside of the lipid vesicular core isstructured such that it faces a lumen, in which a therapeuticallyeffective compound can be encapsulated. Lipid vesicle structures aresubstantially spherical structures usually made of materials having highamphiphilic lipid content. The lipids of these spherical vesicles arepreferably organized in a lipid layer, more preferably in lipidbilayers, which encapsulate a volume, preferably an aqueous volume. Thisvolume provides a lumen, in which a therapeutically effective compound(e.g. water soluble compound) can be encapsulated. A therapeuticallyeffective compound (e.g. water insoluble compound) can also be comprisedin the lipid layer, particularly lipid bilayers, of said sphericalvesicles, or a therapeutically effective compound (e.g. amphiphiliccompound) can be comprised at the interface between the lipid layer,particularly lipid bilayers, and the encapsulated volume, preferablyaqueous volume, of said spherical vesicles.

The term “positively charged lipid vesicular core” means that the netcharge of the lipid vesicular core is positive. It is preferred that thelipids forming the lipid vesicular core comprise at least one cationiclipid.

The term “encapsulated” in the expression “a therapeutically effectivecompound encapsulated within the vesicular core” refers to the positionof the therapeutically effective compound in the particle and means thatthe therapeutically effective compound is comprised in the vesicularcore, particularly covered by the vesicular core. For example, thetherapeutically effective compound (e.g. water soluble compound) can becomprised in the encapsulated volume, preferably aqueous volume, of thevesicular core, the therapeutically effective compound (e.g. waterinsoluble compound) can be comprised in a lipid layer, particularlylipid bilayers, of the vesicular core, or the therapeutically effectivecompound (e.g. amphiphilic compound) can be comprised at the interfacebetween a lipid layer, particularly lipid bilayers, and the encapsulatedvolume, preferably aqueous volume, of the vesicular core. In all cases,the therapeutically effective compound is encapsulated within thevesicular core.

According to the present invention, the term “lipid” refers to any fattyacid derivative or other amphiphilic compound which is capable offorming a lipid vesicular core. In particular, the term “lipid” refersto any fatty acid derivative which is capable of forming a bilayer suchthat a hydrophobic part of the lipid molecule orients toward the bilayerwhile a hydrophilic part orients toward the aqueous phase. The term“lipid” comprises neutral, anionic or cationic lipids. Lipids preferablycomprise a hydrophobic domain with at least one, preferably two, alkylchains or a cholesterol moiety and a polar headgroup. The alkyl chainsof the fatty acids in the hydrophobic domain of the lipid are notlimited to a specific length or number of double bonds. Nevertheless, itis preferred that the fatty acid has a length of 10 to 30, preferably 14to 25 carbon atoms. The lipid may also comprise two different fattyacids.

The lipids may include phospholipids or derivatives thereof,sphingolipids or derivatives thereof, or glycolipids or derivativesthereof. The phospholipids may be glycerophospholipids. Examples of aglycerophospholipid include, without being limited thereto,phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol(DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholineand dimyristoyl phosphatidylcholine (DMPC); phosphatidic acid (PA),phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin(SM) and derivatives of the same.

The term “cationic lipid” refers to a lipid having a net positivecharge. The cationic lipid preferably comprises a cationic, meaningpositively charged, headgroup. If the positively charged lipid vesicularcore comprises a cationic lipid, the positively charged headgroup may belocalized outside and inside of the lipid vesicular core. Thus, thepositive charges of the cationic lipids forming the positively chargedlipid vesicular core preferably face the RNA and the therapeuticallyeffective compound. The hydrophobic domain of cationic lipids ispreferably not different from neutral or anionic lipids. The polarheadgroup of the cationic lipids preferably comprises amine derivativessuch as primary, secondary, and/or tertiary amines, quaternary ammonium,various combinations of amines, amidinium salts, or guanidine and/orimidazole groups as well as pyridinium, piperizine and amino acidheadgroups such as lysine, arginine, ornithine and/or tryptophan. Morepreferably, the polar headgroup of the cationic lipid comprises aminederivatives. Most preferably, the polar headgroup of the cationic lipidcomprises a quaternary ammonium. The headgroup of the cationic lipid maycomprise multiple cationic charges. It is preferred, that the headgroupof the cationic lipid comprises one cationic charge. Monocationic lipidsinclude 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or1,2-dioleoyl-3-trimethylammonium propane (DOTAP),1,2-dimyristoyl-3-trimethylammonium propane (DMTAP),2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide(DMRIE), didodecyl(dimethyl)azanium bromide (DDAB),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) or3β-[N-(N\-N-dimethylarnino-ethane)carbamoyl]cholesterol (DC-Chol), butare not limited thereto. The cationic lipids may be used alone or incombination with cholesterol, with neutral phospholipids or other knownlipid assembly components. The positively charged lipid vesicular coremay also include other components typically used in the formation ofvesicles (e.g. for stabilization). Examples of such other componentsincludes, without being limited thereto, fatty alcohols, fatty acids,and/or cholesterol esters or any other pharmaceutically acceptableexcipients which may affect the surface charge, the membrane fluidityand assist in the incorporation of the lipid into the lipid assembly.Examples of sterols include cholesterol, cholesteryl hemisuccinate,cholesteryl sulfate, or any other derivatives of cholesterol.Preferably, the at least one cationic lipid comprises DMEPC and/orDOTMA.

It is preferred that the portion of the at least one cationic lipid inthe lipid vesicular core of the particles of the present inventionamounts to at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or 100%. For example,the portion of DOTMA in the lipid vesicular core of the particles of thepresent invention may amount to about 10%, about 20%, about 30%, about40%, or about 50%.

The term “lipid bilayer” refers to a double layer structure of lipids.The term encompasses bilayers of all geometries including but notlimited to planar, curved or spherical bilayers. Preferably, thepositively charged lipid vesicular core comprises a lipid bilayer.

The term “liposome” refers to a vesicle comprising a lipid bilayermembrane. Liposomes comprise a liquid inner volume, preferably anaqueous inner volume. The lipid membrane of the liposome may comprisecomponents such as, but not limited to, fats, oils, waxes, cholesterol,sterols, monoglycerides, diglycerides, phospholipids, glycolipids,steroids, proteins, and other membrane-associated components.Preferably, the lipid vesicular core such as the positively chargedlipid vesicular core is a liposome.

When the lipid vesicular core is a liposome, the liposome may be in theform of multilamellar vesicles (MLV), large unilamellar vesicles (LUV),small unilamellar vesicles (SUV) or multivesicular vesicles (MW) as wellas in other bilayered forms known in the art. The size and lamellarityof the liposome will depend on the manner of preparation and theselection of the type of vesicles to be used will depend on thepreferred mode of administration. For systemic therapeutic purposes,liposomes having a diameter of between 50 and 150 nm are preferred (LUVor SUV). For local treatment, liposomes having larger diameters, such asMLV or MW, can be used.

The liposome may be further modified, for example, by an antibody,preferably recognizing an antigen specifically expressed on the targetcell structure and thereby improving the targeting of the liposome. Theliposome is preferably suitable for transporting negatively chargedmolecules and for transfecting animal cells, preferably mammalian cells,most preferably human cells.

The term “helper lipid” refers to a lipid capable of increasing theeffectiveness of delivery of lipid-based particles such as cationiclipid-based particles to a target, preferably into a cell. The helperlipid can be neutral, positively charged, or negatively charged.Preferably, the helper lipid is neutral or negatively charged. Examplesfor helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine(DOPE), cholesterol (Chol),1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin (POPC) and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), but are not limitedthereto. Preferably, the at least one helper lipid comprises DSPC, DOPE,and/or Chol.

In one embodiment, the at least one cationic lipid comprises DOTMA andthe at least one helper lipid comprises CHOL and POPC or the at leastone helper lipid comprises CHOL, wherein the lipids of the lipidvesicular core of the particles of the present invention are composed ofDOTMA/CHOL/POPC 10/50/40, DOTMA/CHOL/POPC 20/50/30, DOTMA/CHOL/POPC30/50/20, DOTMA/CHOL/POPC 40/50/10, and DOTMA/CHOL/POPC 50/50/0 molarratio, respectively.

The term “ethanol injection technique” refers to a process, in which anethanol solution comprising lipids is rapidly dropped into an aqueoussolution through a needle. This action disperses the lipids throughoutthe solution and promotes lipid vesicular core formation such asliposome formation.

The term “reverse phase evaporation technique” refers to a process, inwhich an organic solution comprising lipids is introduced into anaqueous solution such that a water/oil (w/o) emulsion is created. Thus,the organic solution and the aqueous solution should be immiscible. Theorganic solution is then removed from the water/oil emulsion, e.g. byevaporation. This process leads to lipid vesicular core formation suchas liposome formation. The resulting solution can be further dilutedwith an aqueous solution in order to promote lipid vesicular coreformation such as liposome formation.

Using the ethanol injection technique, the lipid vesicular core such asthe positively charged lipid vesicular core having a therapeuticeffective compound encapsulated therein is preferably formed as follows:an ethanol solution comprising lipids, such as cationic lipids likeDMEPC, DOTMA and/or DOTAP, is injected into an aqueous solutioncomprising a therapeutically effective compound, e.g. a bisphosphonate,particularly aminobisphosphonate like zoledronic acid, e.g. understirring.

Using the reverse phase evaporation technique, the lipid vesicular coresuch as the positively charged lipid vesicular core having a therapeuticeffective compound encapsulated therein is preferably formed as follows:an aqueous solution comprising a therapeutically effective compound,e.g. a bisphosphonate, particularly aminobisphosphonate like zoledronicacid, is introduced into a mixture of lipids, such as cationic lipidslike DMEPC, DOTMA and/or DOTAP, and an organic solvent. The abovecomponents are mixed or agitated, e.g. by sonication, so that a w/oemulsion is formed. Subsequently, the organic solvent is removed fromthe w/o emulsion, e.g. by evaporation. To support liposome formation, anaqueous solution may be added to the resulting solution for dilution.

The particles of the present invention are obtainable by adding RNA tothe vesicular core such as the lipid vesicular core, e.g. the positivelycharged lipid vesicular core having the therapeutically effectivecompound encapsulated therein. In one embodiment, the particles of thepresent invention are obtainable by a process comprising a step ofextruding and/or a step of lyophilizing the particle. Preferably, theparticles are extruded, e.g. by filtration, trough a membrane havingpores with a diameter of 0.02 to 1 μm, preferably of 0.3 to 0.6 μm orbetween 0.02 and 0.2 μm. It is to be understood, that the size of thepores are chosen in dependence of the desired size of the particles. Itis preferred, that the membrane is a polycarbonate membrane or celluloseester membrane. The not encapsulated therapeutically effective compoundis preferably removed via dialysis.

The term “extruding” or “extrusion” refers to the creation of objectssuch as particles having a fixed, cross-sectional profile. Inparticular, it refers to the downsizing of a particle, preferably aliposome, whereby the particle is forced through filters with definedpores.

The term “lyophilizing” or “lyophilization” refers to the freeze-dryingof a particle by freezing it and then reducing the surrounding pressureto allow the frozen medium in the particle to sublimate directly fromthe solid phase to the gas phase.

The term “therapeutically effective compound” relates to any compoundbeing therapeutically effective when administered to an individual. Theterm “therapeutically effective compound” further relates to any agentthat changes, preferably cures, alleviates or partially arrests theclinical manifestations of a given disease and its complications in atherapeutic intervention comprising the administration of said compound.

In one embodiment, the therapeutic effective compound encapsulatedwithin the vesicular core of the particles of the present invention iswater-soluble. Hydrophilic properties of the therapeutic effectivecompound may improve its encapsulating efficiency and prevent undesiredrelease. It is preferred, that the therapeutic effective compound has anet negative charge. It is more preferred that the therapeutic effectivecompound is double negatively charged. In one embodiment, thetherapeutically effective compound is a small molecule. A small size ofthe compound may further improve the encapsulating efficiency. Smallmolecule compounds are described to act as good antagonist, agonists orallosteric modulators of diverse targets.

In the present context, the term “treatment”, “treating” or “therapeuticintervention” relates to the management and care of an individual forthe purpose of combating a condition such as a disease or disorder. Theterm is intended to include the full spectrum of treatments for a givencondition from which the individual is suffering, such as administrationof the therapeutically effective compound to alleviate the symptoms orcomplications, to delay the progression of the disease, disorder orcondition, to alleviate or relief the symptoms and complications, and/orto cure or eliminate the disease, disorder or condition as well as toprevent the condition, wherein prevention is to be understood as themanagement and care of an individual for the purpose of combating thedisease, condition or disorder and includes the administration of theactive compounds to prevent the onset of the symptoms or complications.The individual to be treated is an animal, preferably a mammal, inparticular a human being. In the present context, the term“therapeutically effective amount” of a compound means an amountsufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and its complications in a therapeuticintervention comprising the administration of a compound. The desiredreaction for a therapy of a disease or a condition may also be theretardation of the occurrence or the inhibition of the occurrence of thedisease or the condition. An therapeutically effective amount of acompound according to the present invention is dependent on thecondition or disease, the severity of the disease, the individualparameters of the individual, including age, physiological condition,height, and weight, the duration of the treatment, the type of anoptionally accompanying therapy, the specific administration route, andsimilar factors.

Terms such as “RNA forming a hydrophilic shell” or “RNA decorating”according to the invention mean that at least one RNA molecule ispositioned on the outside of a vesicular core. Preferably, the RNA doesnot substantially intercalate into the vesicular core. Preferably, aportion or the entire surface of the vesicular core is covered by theRNA. For example, at least 5%, at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100% of the surface of the vesicular core is covered bythe RNA.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues. The term“ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a β-D-ribofuranosylgroup. The term “RNA” comprisesdouble-stranded RNA, single stranded RNA, isolated RNA such as partiallyor completely purified RNA, essentially pure RNA, synthetic RNA, andrecombinantly generated RNA such as modified RNA which differs fromnaturally occurring RNA by addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs, particularly analogs of naturally-occurring RNAs. The RNAused according to the present invention may have a known composition, orthe composition of the RNA may be partially or entirely unknown. Theterm “mRNA” means “messenger-RNA” and relates to a transcript which isgenerated by using a DNA template and encodes a peptide or protein.Typically, mRNA comprises a 5′-UTR, a protein coding region, and a3′-UTR. mRNA may be generated by in vitro transcription from a DNAtemplate. The in vitro transcription methodology is known to the skilledperson. For example, there is a variety of in vitro transcription kitscommercially available. The term “antisense-RNA” relates tosingle-stranded RNA comprising ribonucleotide residues, which arecomplementary to the mRNA. The term “siRNA” means “small interferingRNA”, which is a class of double-stranded RNA-molecules preferablycomprising 20 to 25 base pairs. Preferably, siRNA is capable of bindingspecifically to a portion of the mRNA-molecule. This binding induces aprocess, in which the said portion of the mRNA-molecule is cut andthereby the gene expression of said mRNA-molecule inhibited. The term“microRNA” refers to a non-coding single-stranded RNA moleculepreferably comprising 20 to 25 base pairs. Preferably, microRNA iscapable of binding specifically to a portion of the mRNA-molecule. Thisbinding induces a process, in which the translation of the said mRNAmolecule and thereby the gene expression of said mRNA molecule isinhibited. The RNA may be modified by a 5′-cap or 5′-cap analog, e.g.achieved by in vitro transcription of a DNA template in presence of said5′-cap or 5′-cap analog, wherein said 5′-cap is co-transcriptionallyincorporated into the generated RNA strand, or the RNA may be generated,for example, by in vitro transcription, and the 5′-cap may be attachedto the RNA post-transcriptionally using capping enzymes, for example,capping enzymes of vaccinia virus. The RNA may comprise furthermodifications. For example, a further modification of the RNA used inthe present invention may be an extension or truncation of the naturallyoccurring poly(A) tail or an alteration of the 5′- or 3′-untranslatedregions (UTR) such as introduction of a UTR which is not related to thecoding region of said RNA.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”. According to the presentinvention, the RNA used in the present invention preferably is in vitrotranscribed RNA (IVT-RNA) and may be obtained by in vitro transcriptionof an appropriate DNA template. The promoter for controllingtranscription can be any promoter for any RNA polymerase. A DNA templatefor in vitro transcription may be obtained by cloning of a nucleic acid,in particular cDNA, and introducing it into an appropriate vector for invitro transcription. The cDNA may be obtained by reverse transcriptionof RNA.

The cDNA containing vector template may comprise vectors carryingdifferent cDNA inserts which following transcription results in apopulation of different RNA molecules optionally capable of expressingdifferent peptides or proteins or may comprise vectors carrying only onespecies of cDNA insert which following transcription only results in apopulation of one RNA species capable of expressing only one peptide orprotein. Thus, it is possible to produce RNA capable of expressing asingle peptide or protein only or to produce compositions of differentRNAs capable of expressing more than one peptide or protein, e.g. acomposition of peptides or proteins.

The term “expression” is used herein in its broadest meaning andcomprises the production of RNA or of RNA and protein. With respect toRNA, the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. Expression may be transient or maybe stable.

According to the present invention, the RNA can be coding RNA, i.e. RNAencoding a peptide or protein. Said RNA may express the encoded peptideor protein. For example, said RNA may be RNA encoding and expressing anantigen or an immunologically active compound (which does not encode anantigen). Alternatively, the RNA can be non-coding RNA such asantisense-RNA, micro RNA (miRNA) or siRNA.

According to the invention, RNA forming a hydrophilic shell on at leasta portion of a vesicular core preferably comprises or consists ofpharmaceutically active RNA.

A “pharmaceutically active RNA” is a RNA that encodes a pharmaceuticallyactive peptide or protein or is pharmaceutically active in its own,e.g., it has one or more pharmaceutical activities such as thosedescribed for pharmaceutically active proteins. For example, the RNA maybe one or more strands of RNA interference (RNAi). Such agents includeshort interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs), orprecursor of a siRNA or microRNA-like RNA, targeted to a targettranscript, e.g., a transcript of an endogenous disease-relatedtranscript of a subject.

A “pharmaceutically active peptide or protein” has a positive oradvantageous effect on the condition or disease state of a subject whenadministered to the subject in a therapeutically effective amount.Preferably, a pharmaceutically active peptide or protein has curative orpalliative properties and may be administered to ameliorate, relieve,alleviate, reverse, delay onset of or lessen the severity of one or moresymptoms of a disease or disorder. A pharmaceutically active peptide orprotein may have prophylactic properties and may be used to delay theonset of a disease or to lessen the severity of such disease orpathological condition. The term “pharmaceutically active peptide orprotein” includes entire proteins or polypeptides, and can also refer topharmaceutically active fragments thereof. It can also includepharmaceutically active analogs of a peptide or protein. The term“pharmaceutically active peptide or protein” includes peptides andproteins that are antigens, i.e., administration of the peptide orprotein to a subject elicits an immune response in a subject which maybe therapeutic or partially or fully protective.

Examples of pharmaceutically active proteins include, but are notlimited to, cytokines and immune system proteins such as immunologicallyactive compounds (e.g., interleukins, colony stimulating factor (CSF),granulocyte colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), erythropoietin, tumor necrosisfactor (TNF), interferons, integrins, addressins, seletins, homingreceptors, T cell receptors, immunoglobulins, soluble majorhistocompatibility complex antigens, immunologically active antigenssuch as bacterial, parasitic, or viral antigens, allergens,autoantigens, antibodies), hormones (insulin, thyroid hormone,catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin,dopamine, bovine somatotropin, leptins and the like), growth hormones(e.g., human grown hormone), growth factors (e.g., epidermal growthfactor, nerve growth factor, insulin-like growth factor and the like),growth factor receptors, enzymes (tissue plasminogen activator,streptokinase, cholesterol biosynthestic or degradative, steriodogenicenzymes, kinases, phosphodiesterases, methylases, de-methylases,dehydrogenases, cellulases, proteases, lipases, phospholipases,aromatases, cytochromes, adenylate or guanylaste cyclases, neuramidasesand the like), receptors (steroid hormone receptors, peptide receptors),binding proteins (growth hormone or growth factor binding proteins andthe like), transcription and translation factors, tumor growthsuppressing proteins (e.g., proteins which inhibit angiogenesis),structural proteins (such as collagen, fibroin, fibrinogen, elastin,tubulin, actin, and myosin), blood proteins (thrombin, serum albumin,Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissueplasminogen activator, protein C, von Wilebrand factor, antithrombinIII, glucocerebrosidase, erythropoietin granulocyte colony stimulatingfactor (GCSF) or modified Factor VIII, anticoagulants and the like.

In one embodiment, the pharmaceutically active protein according to theinvention is a cytokine which is involved in regulating lymphoidhomeostasis, preferably a cytokine which is involved in and preferablyinduces or enhances development, priming, expansion, differentiationand/or survival of T cells. In one embodiment, the cytokine is aninterleukin. In one embodiment, the pharmaceutically active proteinaccording to the invention is an interleukin selected from the groupconsisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

In one particularly preferred embodiment of the invention, the RNAforming a hydrophilic shell on at least a portion of a vesicular corecomprises RNA that encodes a cytokine which is involved in andpreferably induces or enhances development, priming, expansion,differentiation and/or survival of T cells, preferably an interleukinsuch as an interleukin selected from the group consisting of IL-2, IL-7,IL-12, IL-15, and IL-21, and the at least one therapeutically effectivecompound encapsulated within the vesicular core comprises an agentstimulating γδ T cells such as zoledronic acid.

According to the present invention, the term “peptide” comprises oligo-and polypeptides and refers to substances comprising two or more,preferably three or more, preferably four or more, preferably six ormore, preferably eight or more, preferably ten or more, preferably 14 ormore, preferably 16 or more, preferably 21 or more and up to preferably8, 10, 20, 30, 40, or 50, in particular 100 amino acids joint covalentlyby peptide bonds. The term “protein” refers to large peptides,preferably to peptides with more than 100 amino acid residues, but ingeneral the terms “peptides” and “proteins” are synonymous and are usedinterchangeably herein.

According to the invention, the term “RNA encoding” means that the RNA,if present in the appropriate environment, preferably within a cell, candirect the assembly of amino acids to produce the protein or peptide isencodes during the process of translation. Preferably, RNA according tothe invention is able to interact with the cellular translationmachinery allowing translation of the protein or peptide.

According to the present invention, the RNA is preferably negativelycharged and is capable of forming complexes with cationic lipids and, inparticular, covering the surface or portions of a positively chargedlipid vesicular core such as a liposome comprising cationic lipids.

The term “net charge of the particle” relates to the total sum ofcharges, such as positive and negative charges. For example, if theparticle comprises a higher number of negative charges than positivecharges, the net charge of the particle is negative. If the particlecomprises a higher number of positive charges than negative charges, thenet charge of the particle is positive. If the particle comprises anequal number of positive charges and negative charges, the net charge ofthe particle is neutral, particularly electroneutral. Thus, the netcharge of the particle according to the present invention can benegative, positive or neutral. Preferably, the net charge of theparticle is negative.

The term “average diameter” refers to the mean diameter of the particlesand may be calculated by dividing the sum of the diameter of eachparticle by the total number of particles. Although the term “diameter”is used normally to refer to the maximal length of a line segmentpassing through the centre and connecting two points on the periphery ofa spherical object, it is also used herein to refer to the maximallength of a line segment passing through the center and connecting twopoints on the periphery of particles having a substantial sphericalshape or other shapes.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response is to be generated. The term “antigen” includesin particular proteins, peptides, polysaccharides, nucleic acids,especially RNA and DNA, and nucleotides. The term “antigen” alsoincludes agents, which become antigenic—and sensitizing—only throughtransformation (e.g. intermediately in the molecule or by completionwith body protein). An antigen is preferably presentable by cells of theimmune system such as antigen presenting cells like dendritic cells ormacrophages. In addition, an antigen or a processing product thereof ispreferably recognizable by a T or B cell receptor, or by animmunoglobulin molecule such as an antibody. In a preferred embodiment,the antigen is a disease-associated antigen, such as a tumor antigen, aviral antigen, or a bacterial antigen.

The term “tumor antigen” refers to a constituent of cancer cells whichmay be derived from the cytoplasm, the cell surface and the cellnucleus. In particular, it refers to those antigens which are produced,preferably in large quantity, intracellularly or as surface antigens ontumor cells. Examples for tumor antigens include HER2, EGFR, VEGF,CAMPATH1-antigen, CD22, CA-125, HLA-DR, Hodgkin-lymphoma or mucin-1, butare not limited thereto.

The term “viral antigen” refers to any viral component having antigenicproperties, i.e. being able to provoke an immune response in anindividual. The viral antigen may be a viral ribonucleoprotein or anenvelope protein.

The term “bacterial antigen” refers to any bacterial component havingantigenic properties, i.e. being able to provoke an immune response inan individual. The bacterial antigen may be derived from the cell wallor cytoplasm membrane of the bacterium.

The term “disease-associated antigen” is used in it broadest sense torefer to any antigen associated with a disease. A disease-associatedantigen is a molecule which contains epitopes that will stimulate ahost's immune system to make a cellular antigen-specific immune responseand/or a humoral antibody response against the disease. Thedisease-associated antigen may therefore be used for therapeuticpurposes. Disease-associated antigens are preferably associated withinfection by microbes, typically microbial antigens, or associated withcancer, typically tumors.

The term “disease” refers to an abnormal condition that affects the bodyof an individual. A disease is often construed as a medical conditionassociated with specific symptoms and signs. A disease may be caused byfactors originally from an external source, such as infectious disease,or it may be caused by internal dysfunctions, such as autoimmunediseases. In humans, “disease” is often used more broadly to refer toany condition that causes pain, dysfunction, distress, social problems,or death to the individual afflicted, or similar problems for those incontact with the individual. In this broader sense, it sometimesincludes injuries, disabilities, disorders, syndromes, infections,isolated symptoms, deviant behaviors, and atypical variations ofstructure and function, while in other contexts and for other purposesthese may be considered distinguishable categories. Diseases usuallyaffect individuals not only physically, but also emotionally, ascontracting and living with many diseases can alter one's perspective onlife, and one's personality.

The term “disease involving an antigen” refers to any disease whichimplicates an antigen, e.g. a disease which is characterized by thepresence of an antigen. The disease involving an antigen can be aninfectious disease, an autoimmune disease, or a cancer disease or simplycancer. As mentioned above, the antigen may be a disease-associatedantigen, such as a tumor-associated antigen, a viral antigen, or abacterial antigen.

The term “infectious disease” refers to any disease which can betransmitted from individual to individual or from organism to organism,and is caused by a microbial agent (e.g. common cold). Infectiousdiseases are known in the art and include, for example, a viral disease,a bacterial disease, or a parasitic disease, which diseases are causedby a virus, a bacterium, and a parasite, respectively. In this regard,the infectious disease can be, for example, hepatitis, sexuallytransmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis,HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B,hepatitis C, cholera, severe acute respiratory syndrome (SARS), the birdflu, and influenza.

The term “autoimmune disease” refers to any disease in which the bodyproduces an immunogenic (i.e. immune system) response to someconstituent of its own tissue. In other words, the immune system losesits ability to recognize some tissue or system within the body as selfand targets and attacks it as if it were foreign. Autoimmune diseasescan be classified into those in which predominantly one organ isaffected (e.g. hemolytic anemia and anti-immune thyroiditis), and thosein which the autoimmune disease process is diffused through many tissues(e.g. systemic lupus erythematosus). For example, multiple sclerosis isthought to be caused by T cells attacking the sheaths that surround thenerve fibers of the brain and spinal cord. This results in loss ofcoordination, weakness, and blurred vision. Autoimmune diseases areknown in the art and include, for instance, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma,psoriasis, and the like.

The terms “cancer disease” or “cancer” refer to or describe thephysiological condition in an individual that is typically characterizedby unregulated cell growth. Examples of cancers include, but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticularly, examples of such cancers include bone cancer, blood cancerlung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of thehead or neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer, coloncancer, breast cancer, prostate cancer, uterine cancer, carcinoma of thesexual and reproductive organs, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of thebladder, cancer of the kidney, renal cell carcinoma, carcinoma of therenal pelvis, neoplasms of the central nervous system (CNS),neuroectodermal cancer, spinal axis tumors, glioma, meningioma, andpituitary adenoma. The term “cancer” according to the invention alsocomprises cancer metastases.

The term “immune response” relates to a reaction of the immune systemsuch as to immunogenic organisms, such as bacteria or viruses, cells orsubstances. The term “immune response” includes the innate immuneresponse and the adaptive immune response. Preferably, the immuneresponse is related to an activation of immune cells, an induction ofcytokine biosynthesis and/or antibody production.

It is preferred that the immune response induced by the particles of thepresent invention comprises the steps of activation of antigenpresenting cells, such as dendritic cells and/or macrophages,presentation of an antigen or fragment thereof by said antigenpresenting cells and activation of cytotoxic T cells due to thispresentation.

The term “immunologically active compound” relates to any compoundaltering an immune response, preferably by inducing and/or suppressingmaturation of immune cells, inducing and/or suppressing cytokinebiosynthesis, and/or altering humoral immunity by stimulating antibodyproduction by B cells. Immunologically active compounds possess potentimmunostimulating activity including, but not limited to, antiviral andantitumor activity, and can also down-regulate other aspects of theimmune response, for example shifting the immune response away from aTH2 immune response, which is useful for treating a wide range of TH2mediated diseases. Immunologically active compounds can be useful asvaccine adjuvants. In one embodiment, the RNA forming a hydrophilicshell on at least a portion of the vesicular core of the particles ofthe present invention encodes an immunologically active compound. Saidcompound preferably does not encode an antigen.

The term “immune cells” refers to cells of the immune system involved indefending the body of an individual. The term “immune cells” encompassesspecific types of immune cells and their precursors including leucocytescomprising macrophages, monocytes (precursors of macrophages),granulocytes such as neutrophils, eosinophils and basophils, dendriticcells, mast cells, and lymphocytes such as B cells, T cells and naturalkiller (NK) cells. Macrophages, monocytes (precursors of macrophages),neutrophils, dendritic cells, and mast cells are phagocytic cells.

The term “phagocytic cells” refers to cells that protect the body of anindividual by ingesting (phagocytosing) harmful foreign particles,bacteria, and dead or dying cells.

The term “macrophage” refers to a subgroup of phagocytic cells producedby the differentiation of monocytes. Macrophages which are activated byinflammation, immune cytokines or microbial products nonspecificallyengulf and kill foreign pathogens within the macrophage by hydrolyticand oxidative attack resulting in degradation of the pathogen. Peptidesfrom degraded proteins are displayed on the macrophage cell surfacewhere they can be recognized by T cells, and they can directly interactwith antibodies on the B cell surface, resulting in T and B cellactivation and further stimulation of the immune response. Macrophagesbelong to the class of antigen presenting cells. Preferably, themacrophages are splenic macrophages.

The term “dendritic cell” (DC) refers to another subtype of phagocyticcells belonging to the class of antigen presenting cells. Preferably,dendritic cells are derived from hematopoietic bone marrow progenitorcells. These progenitor cells initially transform into immaturedendritic cells. These immature cells are characterized by highphagocytic activity and low T cell activation potential. Immaturedendritic cells constantly sample the surrounding environment forpathogens such as viruses and bacteria. Once they have come into contactwith a presentable antigen, they become activated into mature dendriticcells and begin to migrate to the spleen or to the lymph node. Immaturedendritic cells phagocytose pathogens and degrade their proteins intosmall pieces and upon maturation present those fragments at their cellsurface using MHC molecules. Simultaneously, they upregulatecell-surface receptors that act as co-receptors in T cell activationsuch as CD80, CD86, and CD40 greatly enhancing their ability to activateT cells. They also upregulate CCR7, a chemotactic receptor that inducesthe dendritic cell to travel through the blood stream to the spleen orthrough the lymphatic system to a lymph node. Here they act asantigen-presenting cells and activate helper T cells and killer T cellsas well as B cells by presenting them antigens, alongside non-antigenspecific co-stimulatory signals. Thus, dendritic cells can activelyinduce a T cell- or B cell-related immune response. Preferably, thedendritic cells are splenic dendritic cells.

The term “antigen presenting cell” (APC) is a cell of a variety of cellscapable of displaying, acquiring, and/or presenting at least one antigenor antigenic fragment on (or at) its cell surface. Antigen-presentingcells can be distinguished in professional antigen presenting cells andnon-professional antigen presenting cells.

The term “professional antigen presenting cells” relates to antigenpresenting cells which constitutively express the MajorHistocompatibility Complex class II (MHC class II) molecules requiredfor interaction with naive T cells. If a T cell interacts with the MHCclass II molecule complex on the membrane of the antigen presentingcell, the antigen presenting cell produces a co-stimulatory moleculeinducing activation of the T cell. Professional antigen presenting cellscomprise dendritic cells and macrophages.

The term “non-professional antigen presenting cells” relates to antigenpresenting cells which do not constitutively express MHC class IImolecules, but upon stimulation by certain cytokines such asinterferon-gamma. Exemplary, non-professional antigen presenting cellsinclude fibroblasts, thymic epithelial cells, thyroid epithelial cells,glial cells, pancreatic beta cells or vascular endothelial cells.

The term “maturation” is defined herein as the action of immature highlyphagocytic dendritic cells and macrophages resulting in phenotypicand/or functional modifications of these cells. Especially, in dendriticcells, the associated phenotypic modification is represented by anincrease of CD40, CD80, CD86, CD83, MHC class I and II molecule cellsurface expression and/or a decrease of CD 14 molecule cell surfaceexpression. The functional changes may be the loss of phagocyticproperties, the acquisition of migration abilities, an increasedallogeneic T cell stimulation efficiency and changes in the cytokine andchemokine expression profile, and particularly an increased IL-12secretion. The IL-12 production by DCs is critical for their in vivofunction, since this cytokine has been shown to generate a polarizationof the immune response towards the Thl pathway in vivo. A Thl typeimmune response is considered as immune response involving stimulationof antigen specific T lymphocytes CD8+, whereas a Th2 type immuneresponse involves rather a stimulation of antibody response andeventually unresponsiveness of the cytotoxic lymphocytes to an antigen.

If, according to the present invention, it is desired to induce orenhance an immune response by using particles as described herein, theimmune response may be triggered or enhanced by the therapeuticallyeffective compound encapsulated within the vesicular core. For example,the therapeutically effective compound may stimulate certain immunecells such as T cells. Preferably, said T cells are γδ T cells, morepreferably Vγ9Vδ2 T cells. Alternatively or additionally, the immuneresponse may be triggered or enhanced by the RNA forming a hydrophilicshell on at least a portion of the vesicular core of the particles. Forexample, proteins or peptides encoded by the RNAs or procession productsthereof may be presented by major histocompatibility complex (MHC)proteins expressed on antigen presenting cells. The MHC peptide complexcan then be recognized by immune cells such as T cells or B cellsleading to their activation.

The terms “T-cells” or “T lymphocytes” relate to types of lymphocytesthat play a central role in cell-mediated immunity. T-cells or Tlymphocytes can be distinguished from other lymphocytes, such as B cellsand natural killer (NK) cells, by the presence of a T cell receptor(TCR) on the cell surface. They do not have antigen presentingproperties (but rather, requiring B cells or NK cells for itsantigen-presenting property). They are called T cells because theymature in the thymus. T cells are capable of recognizing an antigen whendisplayed on the surface of antigen presenting cells or matrix togetherwith one or more MHC molecules or one or more non-classical MHCmolecules.

The term “γδ T cells” (gamma delta T cells) relates to a subset of Tcells that possess a distinct T cell receptor (TCR) on their surface. Amajority of T cells have a TCR composed of two glycoprotein chainscalled α- and β-TCR chains. In contrast, in γδ T cells, the TCR is madeup of one γ-chain and one δ-chain. This group of T cells is usually muchless common than αβ T cells. Human γδ T cells play an important role instress-surveillance responses like infectious diseases and autoimmunity.Transformation-induced changes in tumors are also suggested to causestress-surveillance responses mediated by γδ T cells and enhanceantitumor immunity. Importantly, after antigen engagement, activated γδT cells at lesional sites provide cytokines (e.g. INFγ, TNFα) and/orchemokines mediating recruitment of other effector cells and showimmediate effector functions such as cytotoxicity (via death receptorand cytolytic granules pathways) and ADCC.

The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cellreceptor (TCRγδ). The term “Vγ9Nδ2 T cells” relates to cells whichconstitute the major γδ T cell population in human peripheral blood.Vγ9Vδ2 T cells are unique to humans and primates and are assumed to playan early and essential role in sensing “danger” by invading pathogens asthey expand dramatically in many acute infections and may exceed allother lymphocytes within a few days, e.g. in tuberculosis,salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis,toxoplasmosis, and malaria.

γδ T cells respond to small non-peptidic phosphorylated antigens(phosphoantigens) such as pyrophosphates synthesized in bacteria andisopentenyl pyrophosphate (IPP) produced in mammalian cells through themevalonate pathway. Whereas IPP production in normal cells is notsufficient for activation of γδ T cells, dysregulation of the mevalonatepathway in tumor cells leads to accumulation of IPP and γδ T cellactivation. IPPs can also be therapeutically increased byaminobisphosphonates, which inhibit the mevalonate pathway enzymefarnesyl pyrophosphate synthase (FPPS). Among others, zoledronic acid(ZA, zoledronate, Zometa™, Novartis) represents such anaminobiphosphonate, which is already clinically administered to patientsfor the treatment of osteoporosis and metastasic bone disease. Upontreatment of PBMCs in vitro, ZA is taken up especially by monocytes. IPPaccumulates in the monocytes and they differentiate toantigen-presenting cells stimulating development of γδ T cells. In thissetting, the addition of interleukin-2 (IL-2) is preferred as growth andsurvival factor for activated γδ T cells. Finally, certain alkylatedamines have been described to activate Vγ9Vδ2 T cells in vitro, howeveronly at millimolar concentrations.

According to the invention, the term “agent stimulating ₇ 8 T cells”relates to compounds stimulating development of γδ T cells, inparticular Vγ9Vδ2 T cells, in vitro and/or in vivo, in particular byinducing activation and expansion of γδ T cells. Preferably, the termrelates to compounds which in vitro and/or in vivo increase isopentenylpyrophosphate (IPP) produced in mammalian cells, preferably byinhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase(FPPS).

One particular group of compounds stimulating γδ T cells arebisphosphonates, in particular nitrogen-containing bisphosphonates(N-bisphosphonates; aminobisphosphonates). According to the invention,zoledronic acid (INN) or zoledronate (marketed by Novartis under thetrade names Zometa, Zomera, Aclasta and Reclast) is a particularlypreferred bisphosphonate. Zometa is used to prevent skeletal fracturesin patients with cancers such as multiple myeloma and prostate cancer,as well as for treating osteoporosis. It can also be used to treathypercalcemia of malignancy and can be helpful for treating pain frombone metastases.

The terms “stimulating T cells” or “stimulation of T cells” refer to theinduction or activation of a T cell response by a primary signal, suchas by the interaction with an antigen-MHC class II complex through the Tcell antigen receptor. The term also includes the co-stimulation of Tcells, such as through cytokines (e.g. CD80 or CD86 proteins). A T cellis activated if it has received a primary signaling event whichinitiates an immune response by the T cell.

The term “priming T cells” refers to the induction of a first contact ofthe T cell with its specific antigen (e.g. by dendritic cells presentingthe antigen to T cells), which causes the differentiation of the T cellinto an effector T cell (e.g. a cytotoxic T cell or a T helper cell).

The terms “expanding T cells” or “expansion of T cells” refer to theincrease of the number of T cells, preferably T cells specificallyrecognizing an antigen. It is preferred, that the number of T cellsspecifically recognizing an antigen, e.g. an antigen encoded from theRNA decorating the particle of the present invention, or a processionproduct of the antigen increases. The antigen or procession product ofthe antigen is preferably presented in the context of MHC molecules,such as on the surface of antigen presenting cells like dendritic cellsor macrophages.

The term “immunotherapy” relates to the treatment of a disease orcondition by inducing, enhancing, or suppressing an immune response.Immunotherapies designed to elicit or amplify an immune response areclassified as activation immunotherapies, while immunotherapies thatreduce or suppress an immune response are classified as suppressionimmunotherapies. The term “immunotherapy” includes antigen immunizationor antigen vaccination, or tumor immunization or tumor vaccination. Theterm “immunotherapy” also relates to the manipulation of immuneresponses such that inappropriate immune responses are modulated intomore appropriate ones in the context of autoimmune diseases such asrheumatic arthritis, allergies, diabetes or multiple sclerosis.

The terms “immunization” or “vaccination” describe the process ofadministering an antigen to an individual with the purpose of inducingan immune response, for example, for therapeutic or prophylacticreasons.

The term “therapeutic treatment” relates to any treatment which improvesthe health status and/or prolongs (increases) the lifespan of anindividual. Said treatment may eliminate the disease in an individual,arrest or slow the development of a disease in an individual, inhibit orslow the development of a disease in an individual, decrease thefrequency or severity of symptoms in an individual, and/or decrease therecurrence in an individual who currently has or who previously has hada disease.

The terms “prophylactic treatment” or “preventive treatment” relate toany treatment that is intended to prevent a disease from occurring in anindividual. The terms “prophylactic treatment” or “preventive treatment”are used herein interchangeably.

The terms “protect”, “prevent”, “prophylactic”, “preventive”, or“protective” relate to the prevention and/or treatment of the occurrenceand/or the propagation of a disease, e.g. tumor, in an individual. Forexample, a prophylactic administration of an immunotherapy, e.g. byadministering the pharmaceutical composition of the present invention,can protect the receiving individual from the development of a tumor.For example, a therapeutic administration of an immunotherapy, e.g. byadministering the pharmaceutical composition of the present invention,can stop the development of a disease, e.g. lead to the inhibition ofthe progress/growth of a tumor. This comprises the deceleration of theprogress/growth of the tumor, in particular a disruption of theprogression of the tumor, which preferably leads to elimination of thetumor. A therapeutic administration of an immunotherapy may protect theindividual, for example, from the dissemination or metastasis ofexisting tumors.

The term “water-soluble compound” refers to any ionic compound (or salt)which is able to dissolve in water. Generally, the underlying solvationarises because of the attraction between positive and negative chargesof the compound with the partially negative and partially positivecharges of the H₂O-molecules, respectively. Compounds which dissolve inwater are also termed “hydrophilic” (“water-loving”). Water solubility(S_(W)), also known as aqueous solubility, is the maximum amount of asubstance that can dissolve in water at equilibrium at a giventemperature and pressure. Generally, the limited amount is given by thesolubility product. Following the definition of solubility in theEuropean. Pharmacopoeia, “sparingly soluble” means that the approximatevolume of solvent in millilitres per gram of solute is from 30 to 100(at a temperature between 15° C. and 25° C.), “soluble” means that theapproximate volume of solvent in millilitres per gram of solute is from10 to 30 (at a temperature between 15° C. and 25° C.), “freely soluble”means that the approximate volume of solvent in millilitres per gram ofsolute is from 1 to 10 (at a temperature between 15° C. and 25° C.), and“very soluble” means that the approximate volume of solvent inmillilitres per gram of solute is less than 1 (at a temperature between15° C. and 25° C.). For purposes of the present invention, RNA isconsidered a hydrophilic compound and a shell formed by RNA isconsidered a “hydrophilic shell”.

The term “small molecule compound” refers to a molecule that can act toaffect biological processes. Small molecules can include any number oftherapeutic agents presently known and used, or can be small moleculessynthesized in a library of such molecules for the purpose of screeningfor biological function(s). The small molecule compound usually have amolecular weight less than about 5,000 daltons (Da), preferably lessthan about 2,500 Da, more preferably less than 1,000 Da, most preferablyless than about 500 Da. The small molecule compound preferably serves asregulating molecule of biological processes such as an enzyme substrate,an antagonist, or an allosterically activating or an allostericallyinhibiting molecule. It is preferred, that the molecule is capable ofbinding to another molecule, such as a protein, nucleic acid orpolysaccharide, and acting as an effector, altering the activity of theother molecule.

The terms “individual” and “subject” are used herein interchangeably.They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog,cat, cattle, swine, sheep, horse or primate) that can be afflicted withor is susceptible to a disease or disorder (e.g., cancer) but may or maynot have the disease or disorder. In many embodiments, the individual isa human being. Unless otherwise stated, the terms “individual” and“subject” do not denote a particular age, and thus encompass adults,elderlies, children, and newborns. In preferred embodiments of thepresent invention, the “individual” or “subject” is a “patient”.

The term “patient” means an individual or subject for treatment, inparticular a diseased individual or subject, including human beings,non-human primates or another animals, in particular mammals such ascows, horses, pigs, sheeps, goats, dogs, cats or rodents such as miceand rats. In particularly preferred embodiments of the presentinvention, the patient is a human being.

The particles of the present invention may be administered in the formof any suitable pharmaceutical composition. The term “pharmaceuticalcomposition” relates to a formulation comprising a therapeuticallyeffective agent or a salt thereof, preferably together withpharmaceutical excipients such as buffers, preservatives and tonicitymodifiers. Said pharmaceutical composition is useful for treating,preventing, or reducing the severity of a disease or disorder byadministration of said pharmaceutical composition to an individual. Apharmaceutical composition is also known in the art as a pharmaceuticalformulation. The pharmaceutical composition can be administered locallyor systemically, preferably systemically. In the context of the presentinvention, the pharmaceutical composition comprises the particle of theinvention. This particle is therapeutically effective.

The term “systemic administration” refers to the administration of atherapeutically effective agent such that the agent becomes widelydistributed in the body of an individual in significant amounts anddevelops a biological effect. For example, the agent may develop itsdesired effect in the blood and/or reaches its desired site of actionvia the vascular system. Typical systemic routes of administrationinclude administration by introducing the agent directly into thevascular system or oral, pulmonary, or intramuscular administrationwherein the agent is adsorbed, enters the vascular system, and iscarried to one or more desired site(s) of action via the blood.

According to the present invention, it is preferred that the systemicadministration is by parenteral administration. The term “parenteraladministration” refers to administration of a therapeutically effectiveagent such that the agent does not pass the intestine. The term“parenteral administration” includes intravenous administration,subcutaneous administration, intradermal administration or intraarterialadministration but is not limited thereto.

The pharmaceutical compositions of the present invention preferablycomprise at least one adjuvant. The term “adjuvant” relates tocompounds, which when administered in combination with an antigen orantigen peptide to an individual, prolongs or enhances or accelerates animmune response. It is assumed that adjuvants exert their biologicalactivity by one or more mechanisms, including an increase of the surfaceof the antigen, a prolongation of the retention of the antigen in thebody, a retardation of the antigen release, targeting of the antigen tomacrophages, increase of the uptake of the antigen, enhancement ofantigen processing, stimulation of cytokine release, stimulation andactivation of immune cells such as B cells, macrophages, dendriticcells, T cells and unspecific activation of immune cells. Adjuvantscomprise a heterogeneous group of compounds such as oil emulsions (e.g.,Freund's adjuvants), mineral compounds (such as alum), bacterialproducts (such as Bordetella pertussis toxin), or immune-stimulatingcomplexes. Examples for adjuvants include saponins, incomplete Freund'sadjuvants, complete Freund's adjuvants, tocopherol or alum, but are notlimited thereto.

The pharmaceutical composition according to the present invention isgenerally applied in a “pharmaceutically effective amount” and in “apharmaceutically acceptable preparation”.

The term “pharmaceutically acceptable” refers to the non-toxicity of amaterial which does not interact with the action of the active componentof the pharmaceutical composition.

The term “pharmaceutically effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of the treatment of a particular disease, thedesired reaction preferably relates to inhibition of the course of thedisease. This comprises slowing down the progress of the disease and, inparticular, interrupting or reversing the progress of the disease. Thedesired reaction in a treatment of a disease may also be delay of theonset or a prevention of the onset of said disease or said condition. Aneffective amount of the particles or compositions described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of theparticles or compositions described herein may depend on various of suchparameters. In the case that a reaction in a patient is insufficientwith an initial dose, higher doses (or effectively higher doses achievedby a different, more localized route of administration) may be used.

The pharmaceutical compositions of the present invention may containsalts, buffers, preserving agents, carriers and optionally othertherapeutic agents. Preferably, the pharmaceutical compositions of thepresent invention comprise one or more pharmaceutically acceptablecarriers, diluents and/or excipients.

The term “excipient” is intended to indicate all substances in apharmaceutical composition which are not active ingredients such asbinders, lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The term “diluent” relates a diluting and/or thinning agent. Moreover,the term “diluent” includes any one or more of fluid, liquid or solidsuspension and/or mixing media.

The term “carrier” relates to one or more compatible solid or liquidfillers or diluents, which are suitable for an administration to ahuman. The term “carrier” relates to a natural or synthetic organic orinorganic component which is combined with an active component in orderto facilitate the application of the active component. Preferably,carrier components are sterile liquids such as water or oils, includingthose which are derived from mineral oil, animals, or plants, such aspeanut oil, soy bean oil, sesame oil, sunflower oil, etc. Salt solutionsand aqueous dextrose and glycerin solutions may also be used as aqueouscarrier compounds.

Pharmaceutically acceptable carriers or diluents for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaroedit. 1985). Examples of suitable carriers include, for example,magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Examples of suitable diluents include ethanol, glycerol and water.

Pharmaceutical carriers, excipients or diluents can be selected withregard to the intended route of administration and standardpharmaceutical practice. The pharmaceutical compositions of the presentinvention may comprise as, or in addition to, the carrier(s),excipient(s) or diluent(s) any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), and/or solubilising agent(s).Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavoring agents maybe provided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

The terms “reducing” or “inhibiting” or similar phrases relate to theability to cause an overall decrease, preferably of at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50% , atleast 80%, or at least 100% in the level, e.g. expression level,particularly compared to a control. The terms “reduce” or “inhibit” orsimilar phrases include a complete or essentially complete reduction orinhibition, i.e. a reduction or inhibition to zero or essentially zero,particularly compared to a control.

The terms “increasing” or “enhancing” or similar phrases relate to theability to cause an overall increase or enhancement, preferably of atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 80%, or at least 100% in the level, e.g. expressionlevel, particularly compared to a control.

The term “RNA accumulation” refers to the enrichment of RNA in itsbroadest sense. Preferably, the enrichment is a local enrichment in abody, organ, tissue, cell type, cell organelles or cell compartment. Theterm “RNA accumulation” preferably relates to a concentration increaseof at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 80%, or at least 100%. According to the present invention, theterm “RNA accumulation” can mean a concentration increase of the RNAover time in an individual, organ, tissue, cell type, cell organelle orcell compartment (e.g. change of RNA concentration before and aftertreatment) or can refer to concentration differences between differentindividuals, organs, tissues, cell-types, cell organelles or cellcompartments (e.g. RNA concentration difference between lung andspleen).

It is preferred in one embodiment that the RNA decorating the particlesof the present invention, after systemic administration, accumulatesand/or is expressed in the spleen. It is further preferred that no oressentially no RNA accumulation and/or RNA expression is induced by thesystemic administration of the particles of the present invention in thelung and/or liver. Preferably, RNA accumulation and/or RNA expression inthe spleen is at least 5-fold, at least 10-fold, at least 20-fold, atleast 30-fold, at least 40-fold, at least 50-fold the amount of RNAaccumulation and/or RNA expression in the lung.

In one embodiment, the particles of the present invention are targetedto the spleen for activating splenic antigen presenting cells. Thus, itis preferred that after systemic administration of the particles of thepresent invention RNA accumulation and/or RNA expression in antigenpresenting cells occurs. Antigen presenting cells are preferablyprofessional antigen presenting cells or non-professional antigenpresenting cells. More preferably, the professional antigen presentingcells are dendritic cells and/or macrophages, even more preferablysplenic dendritic cells and/or splenic macrophages.

In one preferred embodiment, the systemic administration of theparticles of the present invention results in an increase of theexpression of at least one maturation marker in dendritic cells and/ormacrophages such as splenic dendritic cells and/or splenic macrophages.Preferably, the maturation marker is selected from the group consistingof CD40, CD80, CD86, CD83, MHC class I and II molecules such as HLA-DR.More preferably, the maturation marker is selected from the groupconsisting of CD40, CD86, and MHC class II molecules. Even morepreferably, the maturation marker is selected from the group consistingof CD40, CD86, and HLA-DR.

The term “about” means greater or less than the value or range of valuesstated by 1/10 of the stated values, but is not intended to limit anyvalue or range of values. For instance, a concentration value of about30% means a concentration between 27% and 33%. Each value or range ofvalues preceded by the term “about” is also intended to encompass theembodiment of the stated absolute value or range of values.

As mentioned above, RNA according to the present invention covers atleast a portion of the vesicular core. The term “portion” refers to afraction. With respect to a particular structure such as the surface ofa vesicular core, the term “portion thereof” may designate a continuousor a discontinuous fraction thereof. A portion of the surface of thevesicular core may comprise at least 1%, at least 5%, at least 10%, atleast 20%, at least 30%, preferably at least 40%, preferably at least50%, preferably at least 60%, more preferably at least 70%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably 100% of the surface of the vesicular core. The surfacecoverage with RNA can easily be controlled. It depends, for example, onthe amount of RNA and the amount of positively charged lipids used forthe formation of the particles of the present invention. Thereby, thesurface properties of the particles of the present invention can beinfluenced and, thus, the immune inducing potential or the immunemodulating potential of said particle can be varied.

The agents and compositions provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated).

In particular, treatment of cancer represents a field where combinationstrategies are especially desirable since frequently the combined actionof two, three, four or even more cancer drugs/therapies generatessynergistic effects which are considerably stronger than the impact of amonotherapeutic approach. Thus, in another embodiment of the presentinvention, a cancer treatment using the particles of the invention maybe effectively combined with various other drugs. Among those are e.g.combinations with conventional tumor therapies, multi-epitopestrategies, additional immunotherapy, and treatment approaches targetingangiogenesis or apoptosis (for review see e.g. Andersen et al. 2008:Cancer treatment: the combination of vaccination with other therapies.Cancer Immunology Immunotherapy, 57(11): 1735-1743.) Sequentialadministration of different agents may inhibit cancer cell growth atdifferent check points, while other agents may e.g. inhibitneo-angiogenesis, survival of malignant cells or metastases, potentiallyconverting cancer into a chronic disease. The following list providessome non-limiting examples of anti-cancer drugs and therapies which canbe used in combination with the present invention:

1. Chemotherapy

Chemotherapy is the standard of care for multiple types of cancer. Themost common chemotherapy agents act by killing cells that dividerapidly, one of the main properties of cancer cells. Thus, a combinationwith conventional chemotherapeutic drugs such as e.g. alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumour agents which either affect celldivision or DNA synthesis may significantly improve the therapeuticeffects of the present invention by clearing suppressor cells, reboot ofthe immune system, by rendering tumor cells more susceptible to immunemediated killing, or by additional activation of cells of the immunesystem. A synergistic anti-cancer action of chemotherapeutic andvaccination-based immunotherapeutic drugs has been demonstrated inmultiple studies (see e.g. Quoix et al. 2011: Therapeutic vaccinationwith TG4010 and first-line chemotherapy in advanced non-small-cell lungcancer: a controlled phase 2B trial. Lancet Oncol. 12(12): 1125-33.; seealso Liseth et al. 2010: Combination of intensive chemotherapy andanticancer vaccines in the treatment of human malignancies: thehematological experience. J Biomed Biotechnol. 2010: 6920979; see alsoHirooka et al 2009: A combination therapy of gemcitabine withimmunotherapy for patients with inoperable locally advanced pancreaticcancer. Pancreas 38(3): e69-74). There are hundreds of chemotherapeuticdrugs available which are basically suitable for combination therapies.Some (non-limiting) examples of chemotherapeutic drugs which can becombined with the present invention are carboplatin (Paraplatin),cisplatin (Platinol, Platinol-AQ), cyclophosphamide (Cytoxan, Neosar),docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva),etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinibmesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex, Mexate,Amethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar),sunitinib (Sutent), topotecan (Hycamtin), vincristine (Oncovin, VincasarPFS), and vinblastine (Velban).

2. Surgery

Cancer surgery—an operation to remove the tumor—remains the foundationof cancer treatment. Surgery can be combined with other cancertreatments in order to delete any remaining tumor cells. Combiningsurgical methods with subsequent immunotherapeutic treatment is apromising approach which has been demonstrated countless times.

3. Radiation

Radiation therapy remains an important component of cancer treatmentwith approximately 50% of all cancer patients receiving radiationtherapy during their course of illness. The main goal of radiationtherapy is to deprive cancer cells of their multiplication (celldivision) potential. The types of radiation used to treat cancer arephotons radiation (x-rays and gamma rays) and particle radiations(electron, proton and neutron beams.) There are two ways to deliver theradiation to the location of the cancer. External beam radiation isdelivered from outside the body by aiming high-energy rays (photons,protons or particle radiation) to the location of the tumor. Internalradiation or brachytherapy is delivered from inside the body byradioactive sources, sealed in catheters or seeds directly into thetumor site. Radiation therapy techniques which are applicable incombination with the present invention are e.g. fractionation (radiationtherapy delivered in a fractionated regime, e.g. daily fractions of 1.5to 3 Gy given over several weeks), 3D conformal radiotherapy (3DCRT;delivering radiation to the gross tumor volume), intensity modulatedradiation therapy (IMRT; computer-controlled intensity modulation ofmultiple radiation beams), image guided radiotherapy (IGRT; a techniquecomprising pre-radiotherapy imaging which allows for correction), andstereotactic body radiation therapy (SRBT, delivers very high individualdoses of radiation over only a few treatment fractions). For a radiationtherapy review see Baskar et al. 2012: Cancer and radiation therapy:current advances and future directions. Int. J Med Sci. 9(3): 193-199.

4. Antibodies

Antibodies (preferably monoclonal antibodies) achieve their therapeuticeffect against cancer cells through various mechanisms. They can havedirect effects in producing apoptosis or programmed cell death. They canblock components of signal transduction pathways such as e.g. growthfactor receptors, effectively arresting proliferation of tumor cells. Incells that express monoclonal antibodies, they can bring aboutanti-idiotype antibody formation. Indirect effects include recruitingcells that have cytotoxicity, such as monocytes and macrophages. Thistype of antibody-mediated cell kill is called antibody-dependent cellmediated cytotoxicity (ADCC). Antibodies also bind complement, leadingto direct cell toxicity, known as complement dependent cytotoxicity(CDC). Combining surgical methods with immunotherapeutic drugs ormethods is an successful approach, as e.g. demonstrated in Gadri et al.2009: Synergistic effect of dendritic cell vaccination and anti-CD20antibody treatment in the therapy of murine lymphoma. J Immunother.32(4): 333-40. The following list provides some non-limiting examples ofanti-cancer antibodies and potential antibody targets (in brackets)which can be used in combination with the present invention: Abagovomab(CA-125), Abciximab (CD41), Adecatumumab (EpCAM), Afutuzumab (CD20),Alacizumab pegol (VEGFR2), Altumomab pentetate (CEA), Amatuximab(MORAb-009), Anatumomab mafenatox (TAG-72), Apolizumab (HLA-DR),Arcitumomab (CEA), Bavituximab (phosphatidylserine), Bectumomab (CD22),Belimumab (BAFF), Bevacizumab (VEGF-A), Bivatuzumab mertansine (CD44v6), Blinatumomab (CD19), Brentuximab vedotin (CD30 TNFRSF8), Cantuzumabmertansin (mucin CanAg), Cantuzumab ravtansine (MUC1), Capromabpendetide (prostatic carcinoma cells), Carlumab (CNT0888), Catumaxomab(EpCAM, CD3), Cetuximab (EGFR), Citatuzumab bogatox (EpCAM), Cixutumumab(IGF-1 receptor), Claudiximab (Claudin), Clivatuzumab tetraxetan (MUC1),Conatumumab (TRAIL-R2), Dacetuzumab (CD40), Dalotuzumab (insulin-likegrowth factor I receptor), Denosumab (RANKL), Detumomab (B-lymphomacell), Drozitumab (DR5), Ecromeximab (GD3 ganglioside), Edrecolomab(EpCAM), Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensituximab (NPC-1C), Epratuzumab (CD22), Ertumaxomab (HER2/neu, CD3), Etaracizumab(integrin αvβ3), Farletuzumab (folate receptor 1), FBTA05 (CD20),Ficlatuzumab (SCH 900105), Figitumumab (IGF-1 receptor), Flanvotumab(glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganitumab(IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (IL-1β), Girentuximab(carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB),Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovoma (CA-125),Indatuximab ravtansine (SDC1), Intetumumab (CD51), Inotuzumab ozogamicin(CD22), Ipilimumab (CD152), Iratumumab (CD30), Labetuzumab (CEA),Lexatumumab (TRAIL-R2), Libivirumab (hepatitis B surface antigen),Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Lucatumumab (CD40),Lumiliximab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR),Mepolizumab (IL-5), Milatuzumab (CD74), Mitumomab (GD3 ganglioside),Mogamulizumab (CCR4), Moxetumomab pasudotox (CD22), Nacolomab tafenatox(C242 antigen), Naptumomab estafenatox (5T4), Narnatumab (RON),Necitumumab (EGFR), Nimotuzumab (EGFR), Nivolumab (IgG4), Ofatumumab(CD20), Olaratumab (PDGF-R α), Onartuzumab (human scatter factorreceptor kinase), Oportuzumab monatox (EpCAM), Oregovomab (CA-125),Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumoma(MUC1), Pertuzumab (HER2/neu), Pintumomab (adenocarcinoma antigen),Pritumumab (vimentin), Racotumomab (N-glycolylneuraminic acid),Radretumab (fibronectin extra domain-B), Rafivirumab (rabies virusglycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab(CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrotuzumab(FAP), Siltuximab (IL-6), Tabalumab (BAFF), Tacatuzumab tetraxetan(alpha-fetoprotein), Taplitumomab paptox (CD19), Tenatumomab (tenascinC), Teprotumumab (CD221), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2),TNX-650 (IL-13), Tositumomab (CD20), Trastuzumab (HER2/neu), TRBS07(GD2), Tremelimumab (CTLA-4), Tucotuzumab celmoleukin (EpCAM),Ublituximab (MS4A1), Urelumab (4-1BB), Volociximab (integrin α5β1),Votumumab (tumor antigen CTAA16.88), Zalutumumab (EGFR), Zanolimumab(CD4).

5. Cytokines, Chemokines, Costimulatory Molecules, Fusion Proteins

Combined usage of the pharmaceutical compositions of the presentinvention such as the antigen-coding pharmaceutical compositions of thepresent invention with cytokines, chemokines, costimulatory moleculesand/or fusion proteins thereof to evoke beneficial immune modulation ortumor inhibition effects is another embodiment of the present invention.In order to increase the infiltration of immune cells into the tumor andfacilitate the movement of antigen-presenting cells to tumor-draininglymph nodes, various chemokines with C, CC, CXC and CX3C structuresmight be used. Some of the most promising chemokines are e.g CCR7 andits ligands CCL19 and CCL21, furthermore CCL2, CCL3, CCL5, and CCL16.Other examples are CXCR4, CXCR7 and CXCL12. Furthermore, costimulatoryor regulatory molecules such as e.g. B7 ligands (B7.1 and B7.2) areuseful. Also useful are other cytokines such as e.g. interleukinsespecially (e.g. IL-1 to IL17), interferons (e.g. IFNalpha1 toIFNalpha8, IFNalpha10, IFNalpha13, IFNalpha14, IFNalpha16, IFNalpha17,IFNalpha21, IFNbeta1, IFNW, IFNE1 and IFNK), hematopoietic factors, TGFs(e.g. TGF-α, TGF-β, and other members of the TGF family), finallymembers of the tumor necrosis factor family of receptors and theirligands as well as other stimulatory molecules, comprising but notlimited to 4-1BB, 4-1BB-L, CD137, CD137L, CTLA-4GITR, GITRL, Fas, Fas-L,TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LT.beta.R, RANK, EDAR1, XEDAR,Fn114, Troy/Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40,GITR, GITRL, TACI, BAFF-R, BCMA, RELT, and CD95 (Fas/APO-1),glucocorticoid-induced TNFR-related protein, TNF receptor-relatedapoptosis-mediating protein (TRAMP) and death receptor-6 (DR6).Especially CD40/CD4OL and OX40/OX40L are important targets for combinedimmunotherapy because of their direct impact on T cell survival andproliferation. For a review see Lechner et al. 2011: Chemokines,costimulatory molecules and fusion proteins for the immunotherapy ofsolid tumors. Immunotherapy 3 (11), 1317-1340.

6. Bacterial Treatments

Researchers have been using anaerobic bacteria, such as Clostridiumnovyi, to consume the interior of oxygen-poor tumours. These should thendie when they come in contact with the tumour's oxygenated sides,meaning they would be harmless to the rest of the body. Another strategyis to use anaerobic bacteria that have been transformed with an enzymethat can convert a non-toxic prodrug into a toxic drug. With theproliferation of the bacteria in the necrotic and hypoxic areas of thetumour, the enzyme is expressed solely in the tumour. Thus, asystemically applied prodrug is metabolised to the toxic drug only inthe tumour. This has been demonstrated to be effective with thenonpathogenic anaerobe Clostridium sporogenes.

7. Kinase Inhibitors

Another large group of potential targets for complementary cancertherapy comprises kinase inhibitors, because the growth and survival ofcancer cells is closely interlocked with the deregulation of kinaseactivity. To restore normal kinase activity and therefor reduce tumorgrowth a broad range of inhibitors is in used. The group of targetedkinases comprises receptor tyrosine kinases e.g. BCR-ABL, B-Raf, EGFR,HER-2/ErbB2, IGF-IR, PDGFR-a, PDGFR-β, c-Kit, Flt-4, Flt3, FGFR1, FGFR3,FGFR4, CSF1R, c-Met, RON, c-Ret, ALK, cytoplasmic tyrosine kinases e.g.c-SRC, c-YES, Abl, JAK-2, serine/threonine kinases e.g. ATM, Aurora A &B, CDKs, mTOR, PKCi, PLKs, b-Raf, S6K, STK11/LKB1 and lipid kinases e.g.PI3K, SKI. Small molecule kinase inhibitors are e.g. PHA-739358,Nilotinib, Dasatinib, and PD166326, NSC 743411, Lapatinib (GW-572016),Canertinib (CI-1033), Semaxinib (SU5416), Vatalanib (PTK787/ZK222584),Sutent (SU11248), Sorafenib (BAY 43-9006) and Leflunomide (SU101). Formore information see e.g. Zhang et al. 2009: Targeting cancer with smallmolecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

8. Toll-Like Receptors

The members of the Toll-like receptor (TLRs) family are an importantlink between innate and adaptive immunity and the effect of manyadjuvants rely on the activation of TLRs. A large number of establishedvaccines against cancer incorporate ligands for TLRs for boostingvaccine responses. Besides TLR2, TLR3, TLR4 especially TLR7 and TLR 8have been examined for cancer therapy in passive immunotherapyapproaches. The closely related TLR7 and TLR8 contribute to antitumorresponses by affecting immune cells, tumor cells, and the tumormicroenvironment and may be activated by nucleoside analogue structures.All TLR's have been used as stand-alone immunotherapeutics or cancervaccine adjuvants and may be synergistically combined with theformulations and methods of the present invention. For more informationsee van Duin et al. 2005: Triggering TLR signaling in vaccination.Trends in Immunology, 27(1):49-55.

9. Angiogenesis Inhibitors

In addition to therapies which target immune modulatory receptorsaffected by tumor-mediated escape mechanisms and immune suppressionthere are therapies which target the tumor environment. Angiogenesisinhibitors prevent the extensive growth of blood vessels (angiogenesis)that tumors require to survive. The angiogenesis promoted by tumor cellsto meet their increasing nutrient and oxygen demands for example can beblocked by targeting different molecules. Non-limiting examples ofangiogenesis-mediating molecules or angiogenesis inhibitors which may becombined with the present invention are soluble VEGF (VEGF isoformsVEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptorsNeuropilin-1 and Neuropilin-2) 1 and NRP-1, angiopoietin 2, TSP-1 andTSP-2, angiostatin and related molecules, endostatin, vasostatin,calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2,IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18, prothrombin (kringledomain-2), antithrombin III fragment, prolactin, VEGI, SPARC,osteopontin, maspin, canstatin, proliferin-related protein, restin anddrugs like e.g. bevacizumab, itraconazole, carboxyamidotriazole,TNP-470, CM101, IFN-α, platelet factor-4, suramin, SU5416,thrombospondin, VEGFR antagonists, angiostatic steroids+heparin,cartilage-derived angiogenesis Inhibitory factor, matrixmetalloproteinase inhibitors, 2-methoxyestradiol, tecogalan,tetrathiomolybdate, thalidomide, thrombospondin, prolactina Vβ3inhibitors, linomide, tasquinimod, For review see Schoenfeld and Dranoff2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9):976-81.

10. Small Molecule Targeted Therapy Drugs

Small molecule targeted therapy drugs are generally inhibitors ofenzymatic domains on mutated, overexpressed, or otherwise criticalproteins within the cancer cell. Prominent and non-limiting examples arethe tyrosine kinase inhibitors imatinib (Gleevec/Glivec) and gefitinib(Iressa). The use of small molecules e.g. sunitinib malate and/orsorafenib tosylate targeting some kinases in combination with vaccinesfor cancer therapy is also described in previous patent applicationUS2009004213.

11. Virus-Based Vaccines

There are a number of virus-based cancer vaccines available or underdevelopment which can be used in a combined therapeutic approachtogether with the formulations of the present invention. One advantageof the use of such viral vectors is their intrinsic ability to initiateimmune responses, with inflammatory reactions occurring as a result ofthe viral infection creating the danger signal necessary for immuneactivation. An ideal viral vector should be safe and should notintroduce an anti-vector immune response to allow for boostingantitumour specific responses. Recombinant viruses such as vacciniaviruses, herpes simplex viruses, adenoviruses, adeno-associated viruses,retroviruses and avipox viruses have been used in animal tumour modelsand based on their encouraging results, human clinical trials have beeninitiated. Especially important virus-based vaccines are virus-likeparticles (VLPs), small particles that contain certain proteins from theouter coat of a virus. Virus-like particles do not contain any geneticmaterial from the virus and cannot cause an infection but they can beconstructed to present tumor antigens on their coat. VLPs can be derivedfrom various viruses such as e.g. the hepatitis B virus or other virusfamilies including Parvoviridae (e.g. adeno-associated virus),Retroviridae (e.g. HIV), and Flaviviridae (e.g. Hepatitis C virus). Fora general review see Sorensen and Thompsen 2007: Virus-basedimmunotherapy of cancer: what do we know and where are we going? APMIS115(11):1177-93; virus-like particles against cancer are reviewed inBuonaguro et al. 2011: Developments in virus-like particle-basedvaccines for infectious diseases and cancer. Expert Rev Vaccines10(11):1569-83; and in Guillen et al. 2010: Virus-like particles asvaccine antigens and adjuvants: application to chronic disease, cancerimmunotherapy and infectious disease preventive strategies. Procedia inVaccinology 2 (2), 128-133.

12. Multi-Epitope Strategies

The use of multi epitopes shows promising results for vaccination. Fastsequencing technologies combined with intelligent algorithms systemsallow the exploitation of the tumor mutanome and may provide multiepitopes for individualized vaccines which can be combined with thepresent invention. For more information see 2007: Vaccination ofmetastatic colorectal cancer patients with matured dendritic cellsloaded with multiple major histocompatibility complex class I peptides.J Immunother 30: 762-772; furthermore Castle et al. 2012: Exploiting themutanome for tumor vaccination. Cancer Res 72 (5):1081-91.

13. Adoptive T Cell Transfer

For example, a combination of a tumor antigen vaccination and T celltransfer is described in: Rapoport et al. 2011: Combinationimmunotherapy using adoptive T-cell transfer and tumor antigenvaccination on the basis of hTERT and survivin after ASCT for myeloma.Blood 117(3):788-97.

14. Peptide-Based Target Therapies

Peptides can bind to cell surface receptors or affected extracellularmatrix surrounding the tumor. Radionuclides which are attached to thesepeptides (e.g. RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. Especially oligo- or multimers ofthese binding motifs are of great interest, since this can lead toenhanced tumor specificity and avidity. For non-limiting examples seeYamada 2011: Peptide-based cancer vaccine therapy for prostate cancer,bladder cancer, and malignant glioma. Nihon Rinsho 69(9): 1657-61.

15. Other Therapies

There are numerous other cancer therapies which can be combined with thepresent invention in order to create synergistic effects. Non-limitingexamples are treatments targeting apoptosis, hyperthermia, hormonaltherapy, telomerase therapy, insulin potentiation therapy, gene therapyand photodynamic therapy.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES 1. Materials

1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased fromAvanti Polar Lipids (Alabaster, Ala.). Cholesterol (purity 99%) wasobtained from Sigma (St. Louis, Mo.). Zoledronic acid was obtained fromCHEMOS GmbH (Regenstauf, Germany). RNAses free Phosphate buffer salinewas purchased from Ambion (Darmstadt, Germany). RNAses free water waspurchased from B-Braun (Melsungen, Germany). All of reagents were ofanalytical grade.

2. Methods 2.1 Liposome Preparation by Ethanol Injection Technique andFurther Processing 2.1.1 Preparation of the Liposomes

Liposomes were prepared by a modified ethanol injection techniqueaccording to the following protocol:

-   -   1.5 ml of the aqueous solution of zoledronic acid (3.33 mg/ml)        in PBS pH 7.4 was transferred to 50 ml glass beaker. The beaker        was put on a magnetic stirrer and the solution was stirred at        400 rpm using IKA big squid model stirrer (IKA, Konigswinter        Germany).    -   0.55 ml of the lipid/ethanol solution (total lipid concentration        100 mM) was injected by a syringe (Syring type: Omnifix®-F 1 ml        sterile plastic syringe, B. Braun; Melsungen, Germany, Needle:        fine needle with 27G size) into the zoledronic acid solution        under stirring.    -   After lipid injection, the suspension was stirred for 10        minutes.    -   After 10 minutes, 4 ml of PBS pH 7.4 solution was added to the        liposomes. The liposome dispersion was stirred for 20 minutes.

2.1.2 Filtration of the liposomes

The obtained raw dispersion of the liposomes was passed through aMinisart 0.45 μm CE membrane (Sartorius Stedim Biotech GmbH, Goettingen,Germany).

2.1.3 Dialysis of the Liposomes

Dialysis of the filtered liposomes to remove the free zoledronic acid(non-encapsulated) and ethanol residue was carried out as follows:

Each 1 ml of the liposomes was dialyzed versus 400 ml of PBS by usingregenerated cellulose membrane, RNAses free D-tube dialyzer Maxi, MWCO12-14 KDa (Novagen EMD chemicals Inc., San Diego, Calif., USA). Thedialysis took place at room temperature for 24 hrs and at a stirringspeed of 400 rpm. The liposomes were recovered in sterile falcon tubefor further physicochemical characterizations.

2.2 Liposome preparation by reverse-phase evaporation technique andfurther processing

2.2.1 Preparation of the Liposomes

Liposomes were prepared by reverse-phase evaporation technique accordingto the following protocol:

-   -   34.02 mg DSPC, 22.32 mg DOPE and 23.70 mg DOEPC were weighed in        a round bottom flask.    -   Ten ml of chloroform were pipetted into the round bottom flask        to dissolve the lipids. Using a rotary evaporator, chloroform        was evaporated at 60 rpm and 10 mbar for 1 h.    -   The lipid film was redissolved in 3 ml diethyl ether.    -   One ml of zoledronic acid solution (10mg/ml in PBS pH 7.4) was        transferred to the diethylether lipid solution and sonicated for        about two minutes until a homogenous mixture was obtained.    -   The diethylether was removed using a rotary evaporator at 300        mbar and 200 rpm until a homogenous gel was obtained.    -   The vacuum was released and the gel flushed with nitrogen gas.    -   One ml of PBS was added to the gel.    -   The gel was rotated at 200 rpm on the rotary evaporator until a        homogenous suspension of liposomes was obtained.    -   The liposomes were purged from the diethylether traces by        rotation at 60 rpm under high vacuum (200 mbar) for about 2 h.    -   The raw dispersion of the liposomes was transferred to a sterile        15 ml Falcon tube.

2.2.2 Filtration of the Liposomes

The obtained raw dispersion of the liposomes was passed through aMinisart 0.40 μm CE membrane (Sartorius Stedim Biotech GmbH, Goettingen,Germany).

2.2.3 Dialysis of the Liposomes

Dialysis of the filtered liposomes to remove the free zoledronic acid(non-encapsulated) and ethanol residue was carried out as follows:

Each 1 ml of the liposomes was dialyzed versus 400 ml of PBS by usingregenerated cellulose membrane, RNAses free D-tube dialyzer Maxi, MWCO12-14 KDa (Novagen EMD chemicals Inc., San Diego, Calif., USA). Thedialysis took place at room temperature for 24 hrs and at a stirringspeed of 400 rpm. The liposomes were recovered in sterile falcon tubefor further physicochemical characterizations.

2.3 Physicochemical Characterization of the Liposomes 2.3.1 LiposomeSize, Polydispersity Index (PI), and Zeta-Potential

Liposome size, polydispersity index (PI), and zeta-potential wereroutinely measured with a Nicomp 380ZLS laser light scattering particlesizer (Santa Barbara, Calif., USA).

2.3.2 Encapsulated and Free Zoledronic Acid (ZA), and EncapsulationEfficiency Determination

Encapsulated and free zoledronic acid was quantified by HPLC. The HPLCsystem consisted of a G1311B quaternary pump, a G4212B DAD (diode arraydetector) detector, a G1367E auto-sampler AS Hip, a G1330B column oventhermostat, and a ChemStation for LC revision B.04.02 (Agilenttechnologies, Colorado, USA). The stationary phase was xSelect CSH (C18)column (150 mm×4.6 mm×3.5 μm) (Waters, Eschborn, Germany). The mobilephase was a mixture of methanol (20%) and phosphate buffer 30 mM (80%)containing 5 mM tetrabutylammonium bromide (TCI Deutschland GmbH,Eschborn, Germany) adjusted to pH 7.2. An inoLab pH 7310P pH-meter (WTW,Weilheim, Germany) was used for pH determination of the mobile phase.The flow rate and the column oven temperature were 1 mL/min and 50° C.The detection wavelength was 215 nm. The injection volume amounted to 25μl. Free zoledronic acid was determined by using high recovery Ultracelwith a regenerated cellulose membrane and 30 KD MWCO (Millipore,Schwalbach/Ts., Germany) and using the following steps:

-   -   Removal of any preservatives by filtration of 0.1 M NaOH        solution followed by PBS through the membrane.    -   Transfer of 500 p.1 of the liposome sample to 0.5 mL Ultracel        tubes.    -   Centrifugation of the sample at 14000 xg using 40° fixed angle        rotor centrifuge

Pico 21 (Thermoscientific, Osterode, Germany) at room temperature for 15minutes.

-   -   Collection of the filtrate in a HPLC glass vial for        quantification.    -   Measurement of zoledronic acid concentration in the filtrate by        HPLC as mentioned above.

2.3.3 Encapsulation Efficiency % Calculation

Encapsulation efficiency %=[(Total ZA in dialyzed liposomes−Free ZA indialyzed liposomes/Total ZA in undialyzed liposomes (filtered)]×(100).

2.3.4 Formation of ZARNAsomes

According to the required ratio of cationic lipid/RNA (mole/base), thecalculated volume of the ZA-liposomes added to the calculated volume ofRNA/PBS. The mixture incubated for at least 15 minutes to formZARNAsomes.

2.3.5 Determination of Bound RNA/Total RNA in ZARNAsomes

One of the main factors influencing the efficacy of ZARNAsomes is theratio of bound RNA/free RNA. Therefore, it is prerequisite to know howmuch of RNA is bound to ZA-liposomes to form ZARNAsomes. Hence, wedeveloped a new method for quantification of bound/total RNA by usingbioanalyzing technique. The Agilent's 2100 Bioanalyzer works as follows:Charged biomolecules like DNA or RNA are electrophoretically driven by avoltage gradient similar to slab gel electrophoresis. The molecules areseparated by size. Smaller fragments are migrating faster than largerones. Dye molecules intercalate into DNA or RNA strands or Protein-SDSmicelles. These complexes are detected by laser-induced fluorescence.Data are translated into gel-like images (bands) and electropherograms(peaks). With the help of a molecular ladder that contains fragments ofknown sizes and concentrations, a standard curve of migration timeversus fragments size is plotted. From the migration times measured foreach fragment in the sample, the size is calculated. In our experiment,ZARNAsomes were prepared by mixing a calculated volume of ZA-liposomeswith RNA at nine cationic lipid/RNA charge ratios: Cationic lipid/RNA(mole/base)=0.025, 0.125, 0.25, 0.375, 0.50, 0.625, 0.75, 0.875, 1.00.ZARNAsomes were applied to the bioanalyzer chip and free RNA (unbound tolipid) was calculated from standard calibration curve of pure RNAmeasured in the same chip with the samples.

2.4 In Vitro Experiments 2.4.1 Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from Buffy Coatdonations, drawn from the “Transfusions-Zentrale” of UniversitatsmedizinMainz, by density-centrifugation on a Ficoll-Hypaque density gradient.After isolation PBMCs were further processed to generate CD14 positivemononuclear cells by using CD14

MicroBeads and LS columns (Miltenyi Biotec). Purified CD14 positivecells were used to generate conventional immature dendritic cells (iDCs)by 5-day cultivation in standard medium supplemented with GM-CSF (1000U/mL) and IL-4 (1000 U/mL). CD 14-depleted cells, meaning peripheralblood lymphocytes (PBLs), were frozen in liquid nitrogen to besubsequently used in coculture experiments. The standard medium was RPMI1640, containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100mg/mL streptomycin.

2.4.2 Flow Cytometry

The following monoclonal antibodies (mAbs) were used: FITC-labledanti-CD83 (HB15e, BD Pharmingen), PE-labled anti-CD86 (IT2.2, BDPharmingen), APC-labled anti-HLA-DR (G46-6, BD Pharmingen), PacificBlue-labled anti-CD3 (UCHT1, BD Pharmingen), FITC-labled anti-TCR-Vδ2(B6, BD Pharmingen). Viability of cells was always evaluated by usingthe fixable viability dye eFluor506 (eBioscience). All flow cytomericdata were acquired using FACSCanto II Flow cytometer (BD Biosciences)and analysed with FlowJo-Software (Tree Star).

2.4.3 Luc-Assay/RNA Expression

To check if the RNA is still intact when it is bound at the outside ofthe liposomes, the translation of luciferase-encoding RNA was checked bybioluminescence. Here, 2×10⁵ immature dendritic cells (iDCs) were seededin 96-well plate and incubated as indicated for 24 h. After incubation,samples were centrifuged (300 g, 5 min) and supernatants were discarded.For the luciferase assay system (Bright Glo™, Promega), cell pelletswere resuspended in 100 μL standard medium (w/o Pen/Strep) and 1004assay-substrate solution was added to each well. The luminescence wasmeasured after 10 min incubation with a luminescence reader (TecanInfinite M200).

2.4.4 Analysis of Dendritic Cell (DC) Maturation

To evaluate whether formulations/substances lead to a maturation ofdendritic cells (DCs) after incubation, flow cytometry analysis wasperformed. Therefore, immature dendritic cells (iDCs) were seeded instandard medium in a 48-well-Plate (1×10̂6

DCs/mL and well) and incubated as indicated over-night, approximately 20h. After incubation cells were harvested, washed and stained withanti-CD83, anti-CD86 and anti-HLA-DR mABs. As a positive control formaturation, a so-called “maturation cocktail” containing the followingcytokines was used: IL-4 (500 U/mL), GM-CSF (800 U/mL), IL-1β (10ng/mL), TNF-α (10 ng/mL), IL-6 (1000 U/mL) and PGE-2 (1 μg/mL). Foranalysis, the expression of these markers has been normalized tonegative control, meaning no stimulation (cells only in standardmedium).

2.4.5 Vγ9δ2 T Cell Proliferation/Expansion To check the functionality ofthe encapsulated compound, zoledronic acid (ZA), the expansion of Vγ9δ2T cells was evaluated after co-cultivation of ZA-loaded iDCs withcryopreserved PBLs. For evaluation of the ex vivo frequency of Vγ9δ2 Tcells, freshly isolated PBMCs were stained with anti-CD3, anti-TCR-Vδ2mABs and analyzed via flow cytometry. Therefore, iDCs have beenincubated as indicated for 24 h in standard medium (1×10⁶ cells/mL)containing 5 μM ZA. After loading with ZA, iDCs were centrifuged andwashed with PBS. The co-culture of iDCs with PBLs was setup in a ratioof 20 (iDCs) : 1 γ9δ2 T cells ex vivo. The medium was supplemented with10 U/mL IL-2 (Proleukin). After 7-day incubation, cells were harvested,washed and stained with anti-CD3, anti-TCR-Vδ2 mABs to evaluatefrequency of γ982 T cells and their expansion via flow cytometry. Foranalysis, the expansion rate was determined dividing whole cell amountof γ9δ2 T cells before by after 7-day-cultivation.

2.5 In Vivo Experiments 2.5.1 Animals

Female, 6-12 week old Balb/c mice were obtained from in house breedingof the Zentrale Versuchtiereinrichtung (ZVTE) of the Johannes GutenbergUniversity Mainz and housed under normal laboratory conditions withcircadian light/dark cycles and free access to standard mouse chow andtap water (Approval by the Regional Council's Ethics Committee forAnimal Experimentation (Koblenz/Rheinland-Pfalz, Germany, G 12-1-081).Mice were anesthetized with isofluorane and the indicated solutionsinjected retro-orbital.

2.5.2 RNA Expression Analyzed Via Bioluminescence Imaging

Evaluation of uptake and translation of luciferase (Luc) encoding RNAwas performed by non-invasive in vivo bioluminescence imaging using theIVIS Spectrum imaging system (Caliper Life Sciences, Alameda, Calif.,USA). 6 h after injection of indicated solutions mice receivedintraperitoneally an aqueous solution of D-luciferin (150 mg/kg bodyweight). 5 min later photons emitted were collected for 1 min. Measuredbioluminescence signal in regions of interest (ROIs) were quantified andpresented as color-scaled images superimposed on grayscale photos ofmice using the Living Image software (Caliper Life Sciences). Forquantifications, the bioluminescence signal retrieved from therespective organ or tissue was normalized by subtracting backgroundluminescence from a non-signal emitting region.

2.5.3 Splenic DC maturation analyzed via FACS assay

24 h after injection of indicated solutions, mice were euthanized bycervical dislocation and spleens removed. Splenocytes were obtained bydigestion of spleen with collagenase (1 mg/ml; Roche) for 5 min, andsubsequently pressing spleens through a 70 μm nylon cell strainer (BDBiosciences, Heidelberg, Germany) using the plunger of a 1 ml syringe.After washing the mesh with PBS, and a centrifugation step for 5 min at1500 rpm, cells underwent red blood cell lysis (RBC) for 5 min at RTwere the pellet was suspended in hypotonically buffer (KHCO₃/NH₄Cl/EDTA). And after an additional centrifugation step, cells were suspendedin 10 ml PBS/5% FCS. Splenocyte samples were incubated at 4° C. withfluorophore labeled monoclonal antibodies (mAbs) F4-80, CD40, CD86,NK1.1, CD11c, CD8 (all from BD Pharmingen, Heidelberg, Germany) for 30min, washed with PBS and suspended in 300 μl PBS/5% FCS. Flow cytomericdata of 0.75×10⁶ cells were acquired on a FACSCalibur analytic flowcytometer (BD Biosciences) and analyzed with FlowJo (Tree Star)software.

2.5.4 Test of isopentenylpyrophosphate (IPP) accumulation

24 h after injection of indicated solutions, mice were sacrificed andindicated tissue (e.g. spleen) collected. The splenocytes were preparedaccording to the protocol for the splenic DC maturation measurement byFACS assay, without red blood cell lysis. 5×10⁶ splenocytes wereextracted using ice-cold acetonitrile (300 μl) and water (200 μl)containing 0.25 nmol/L NaF and Na₃VO₄ to prevent degradation ofisopentenylpyrophosphate (IPP) (5 min). After centrifugation at 13.000×gfor 1 min, the soluble supernatant extract was transferred to a freshEppendorf tube and dried down in a vacuum centrifuge, then stored at−20° C. until mass spectrometry (MS) analysis of IPP.

2.5.5 Analysis of IPP by Mass Spectrometry

The samples were dissolved in 0.28% (v/v) hexylamine in 2% methanol. Themolar amounts of IPP in cell extracts were determined by an Agilent 1290Infinity UHPLC with a 6490 triple quadrupole mass spectrometer(JetStream Technology, negative ion electrospray ionization). IPP is avery hydrophilic compound and therefore the use of hexylamine as anion-pair agent was necessary to retain this compound into areversed-phase column. HPLC separation was performed using a Poroshell120 EC-C18 column (2.1×50 mm, 2.7 μm) and an eluent system consisting of2.8% (v/v) hexylamine, 1% acetic acid in methanol (1:50 in water, eluentA) and acetonitrile (eluent B). Flow-rate was 0.4 mL/min and injectionvolume 20 μL. After HPLC separation, negative ion mass spectra for IPPwere acquired using a 6490 triple quadrupole mass spectrometer equippedwith an electrospray ionization (ESI) source (Agilent Technologies,Colo., USA). Selected reaction monitoring (SRM) was used for analysis ofthe compounds in the sample and quantitation was based on characteristicfragment ions. The standard curve was created by using synthetic IPP.

The concentrations of the samples were determined using the peak areasof the SRM chromatograms and the standard curve.

3. Results and Discussion

3.1 Zoledronic Acid (ZA) Encapsulated Liposomes Decorated with RNA forImmunotherapy

Zoledronic acid (ZA) encapsulating liposomes (ZA liposomes) withdifferent compositions and molar fractions of the cationic lipid DOTMAwere prepared and the binding of RNA to these liposomes was investigated(see FIG. 1). The liposome composition was as follows: DOTMA/CHOL/POPC10/50/40, DOTMA/CHOL/POPC 20/50/30, DOTMA/CHOL/POPC 30/50/20,DOTMA/CHOL/POPC 40/50/10, and DOTMA/CHOL/POPC 50/50/0 molar ratio,respectively. Thus, the liposomes were composed of 10%, 20%, 30%, 40%,or 50% DOTMA. Binding was investigated by adding an excess of RNA to thezoledronic acid (ZA) encapsulating liposomes (ZA liposomes) andquantifying the RNA by capillary electrophoresis (Bioanalyzer). TheDOTMA/RNA charge ratios were as follows: DOTMA/RNA (mole/base)=0.025,0.125, 0.25, 0.375, 0.50, 0.625, 0.75, 0.875, 1.00. When cationicliposomes were present, the measured amount of RNA decreased. Themissing RNA was taken as liposome bound RNA. As can be seen, the amountof bound RNA was directly proportional to the amount of DOTMA present ina one-to-one stoichiometry with respect to the charge. This means, forall tested liposomes, that the amount of bound RNA was directlycorrelated with the amount of DOTMA in the membrane. As the molarfraction of DOTMA changed (from 10% to 50%), also the amount of boundRNA per liposome and the surface coverage of the liposomes with RNAchanged. Thus, in the given experiment, RNA covered liposomes, where thesurface coverage with RNA changed by a factor of five, could beassembled in a controlled way. From this experiment, it can be concludedthat the amount of RNA decorating the particle of the present inventioncan be regulated by the amount of cationic lipids, e.g. DOTMA, used. Inthis way ZA-liposomes with complete or partial RNA surface coverage canbe generated.

3.2 Particle Sizes and Polydispersity Indices (PIs) of CationicZA-Liposomes After Addition of RNA

After having shown that RNA can be bound to ZA-liposomes in a controlledand efficient way, the properties of the RNA-decorated ZA-liposomes suchas particle size and polydispersity index (PI) were investigated. Thetested liposomes had the following composition: DOTMA/CHOL/POPC30/50/20, molar ratio. FIG. 2 shows the results of the determination ofthe particle size and the polydispersity index (PI) of ZA-liposomescomprising the cationic lipid DOTMA and RNA at different charge ratios(i.e. DOTMA/RNA). An excess of negative charges and an excess ofpositive charges was investigated. In particular, liposomes comprisingthe cationic lipid DOTMA and RNA, wherein the ratio of DOTMA to RNA was1/4, 1/2, 1/1, 2/1, and 4/1+/−, were tested. The sizes andpolydispersity indices (PIs) of the prepared liposomes were measured byphoton correlation spectroscopy. As can be seen in FIG. 2, formulationswith discrete particle sizes between 418 and 563 nm were obtained, whenan excess of negative charges was present (DOTMA to RNA in a ratio of1/4 or 1/2) or when the ratio of DOTMA to RNA was 1 to 1. For thesecharge ratios, no aggregation of the liposomes was observed. The PI ofliposomes with an excess of negative charges was comparable with the PIof pure liposomes. Only moderate changes of the particle size withrespect to the precursor liposomes occurred (see pure liposomesindicated as “Lipo” in FIG. 2) which may be in line with the boundmolecular layer on the liposome surface.

3.3 Particle Size, Polydispersity Index (PI) and Zeta-Potential ofZARNAsomes Prepared by Reverse-Phase Evaporation Technique

ZARNAsomes prepared by reverse-phase evaporation technique were alsocharacterized regarding their particle size, polydispersity index (PI)and zeta-potential. They had a size of 390 nm with a polydispersityindex (PI) of 0.3. The zeta potential was +41 mV. Using HPLC,concentration of zoledronic acid (ZA) in the liposomes was determined.The zoledronic acid concentration was 0.765 mg/ml, while theconcentration of free zoledronic acid was 0.046 mg/ml. Leakage ofliposomes inducing release of zoledronic acid could not be observed.This indicates that both, reverse-phase evaporation technique andethanol injection technique, can be used for preparation of RNAdecorated lipid particles (e.g. ZARNAsomes).

3.4 Expression of Luciferase (Luc) in Vitro Transcribed (IVT) RNA inDendritic Cells (DCs) after incubation with liposome formulations

Next, it was tested, whether RNA coding for a protein such as an antigenand bound to ZA-liposomes was still intact and could be translated intoa functional protein such as antigen in dendritic cells (DCs).Exemplarily, RNA encoding the enzyme luciferase (Luc) was incubated withZA-liposomes. The resulting luciferase RNA decorated ZA-liposomes wereincubated with dendritic cells, and luciferase expression was evaluatedvia luminescence indicating the metabolic rate of luciferin being asubstrate for luciferase in counts per seconds (cps). FIG. 3 shows theresults of the luminescence measurement in dendritic cells incubatedwith ZA-liposomes (ZA-L) (i.e. zoledronic acid (ZA) encapsulatingliposomes), luciferase (Luc) RNA decorated ZA-liposomes (ZARNAsomes)(i.e. luciferase (Luc) RNA decorated zoledronic acid (ZA) encapsulatingliposomes) or naked RNA (i.e. luciferase RNA not bound to ZA-liposomes).As can be seen from FIG. 3, only dendritic cells incubated withluciferase RNA decorated ZA-liposomes (ZARNAsomes) showed a luciferasesignal. This indicates that dendritic cells could take up ZARNAsomeswithout destroying the RNA bound to the ZA-liposomes and that theZARNAsomes were stable enough such that the protein (here luciferase)encoding RNA could be translated. Thus, ZARNAsomes, and accordingly theparticles of the present invention, can be used to induce translation ofa protein such as an antigen of choice in dendritic cells. This furtherindicates, that ZARNAsomes, and accordingly the particles of the presentinvention, can be used for immunotherapy e.g. tumor vaccination.

3.5 Relative Expression of Maturation Markers in Dendritic Cells afterIncubation with Liposome Formulations

FIG. 4 shows the influence of ZARNAsomes (i.e. RNA decorated zoledronicacid (ZA) encapsulating liposomes) on the maturation of dendritic cells(DCs) in vitro compared to the influence of a positive control(maturation cocktail containing IL-4, GM-CSF, IL-1β, TNF-α, IL-6 andPGE-2), naked RNA and ZA-liposomes (ZA-L) (i.e. zoledronic acid (ZA)encapsulating liposomes). To evaluate the maturation of dendritic cellsinduced by ZARNAsomes, the relative expression of the maturation markersCD83, CD86 and HLA-DR was determined using flow cytometry. For analysisof the relative expression, the expression of CD83, CD86 and HLA-DR wasnormalized to the negative control (cells in standard medium). FIG. 4shows that ZARNAsomes resulted in a distinct higher expression of CD83,CD86 and HLA-DR compared to naked RNA and ZA-liposomes (ZA-L). RegardingCD86 and HLA-DR, the expression induced by ZARNAsomes was evencomparable with the positive control. The determined increase of therelative expression induced by ZARNAsomes was between a factor of 1.5and 3 for CD83, between a factor of 2.0 and 3.5 for CD86 and between afactor of 1.5 and 2.0 for HLA-DR. This indicates that ZARNAsomes inducethe maturation of dendritic cells and are, thus, capable of modulatingthe immune response.

3.6 Functionality of Encapsulated Zoledronic Acid (ZA) AfterCo-Cultivation Of Immature Dendritic Cells (iDCs) Incubated withLiposome Formulations and Peripheral Blood Lymphocytes (PBLs)

Next, it was tested, whether an encapsulated therapeutically agent wasstill functional after delivery. Therefore, the functionality ofzoledronic acid (ZA) delivered by the ZARNAsomes was evaluated. Inparticular, the capability of zoledronic acid to induce the expansion ofVγ9Vδ2 T cells was tested (Castella, B., Riganti, C., Fiore, F.,Pantaleoni, F., Canepari, M. E., Peola, S., Foglietta, M., Palumbo, A.,Bosia, A., Coscia, M., Boccadoro, M., Massaia, M. (2011), The Journal ofImmunology 187(4), 1578-90). To investigate the influence of ZARNAsomeson the expansion rate of Vγ9Vδ2 cells, zoledronic acid (ZA) loadedimmature dendritic cells were co-cultured with peripheral bloodlymphocytes containing Vγ9Vδ2 T cells. After seven days of co-culturing,the cells were stained with an anti-CD3 antibody and an anti-TCR-V82antibody to evaluate the frequency of Vγ9Vδ2 cells and their expansionvia flow cytometry. FIG. 5A shows the frequency of Vγ9δ2 T cellsregarding all peripheral blood lymphocytes. As it can be seen,ZARNAsomes (i.e. RNA decorated ZA encapsulating liposomes), ZA-liposomes(ZA-L) (i.e. ZA encapsulating liposomes) and free zoledronic acid (ZA)resulted in an increase of the percentage of Vγ9δ2 T cells compared tothe negative control (no zoledronic acid (ZA)). FIG. 5B shows theexpansion rate of Vγ9δ2 T cells. For analysis, the expansion rate wasdetermined by dividing the whole cell amount of Vγ9δ2 T cells before theseven day cultivation period by the whole cell amount of Vγ9δ2 T cellsafter the seven day cultivation period. As it can be seen, ZARNAsometreatment resulted in an expansion rate of Vγ9δ2 T cells of about60-fold, ZA-liposome (ZA-L) treatment resulted in an expansion rate ofVγ9δ2 T cells of about 40-fold and free zoledronic acid (ZA) treatmentresulted in an expansion rate of Vγ9δ2 T cells of about 15-fold. Thus,ZARNAsome-induced expansion of Vγ9δ2 T cells is highly efficientindicating both high functionality of the encapsulated zoledronic acidand good delivery properties of the zoledronic acid.

3.7 Application of ZARNAsomes Resulted in Luciferase (Luc) Expression inthe Spleen

For validation of the in vitro results in vivo, luciferase (Luc)encoding RNA decorated liposomes, corresponding to 20 μg RNA/ mousewereinjected into Balb/c mice. The translation of luciferase (Luc) RNA boundto liposomes was detected in the presence of luciferin usingbioluminescence imaging. FIG. 6A shows that application of ZARNAsomes(i.e. luciferase (Luc) RNA decorated zoledronic acid (ZA) encapsulatingliposomes) resulted in luciferase expression in the spleen. FIG. 6Afurther shows that injection of ZARNAsomes (i.e. Luciferase (Luc) RNAdecorated zoledronic acid (ZA) encapsulating liposomes) induced asignificant higher luminescence signal than injection of luciferase(Luc) RNA decorated buffer vehicle encapsulating liposomes (EL+Luc-RNA).This indicates that zoledronic acid (ZA) encapsulation does notnegatively influence RNA uptake and translation in vivo. It ratherenhances the expression of protein such as antigen encoding RNA.

3.8 Application of ZARNAsomes Resulted in an Upregulation of CD40 andCD86 Expression on Splenic Dentritic Cells (DCs) and Macrophage (mΦ)Cell Population

Further, it was tested whether injection of ZARNAsomes resulted in thematuration of splenic dendritic cells and macrophages in vivo.Therefore, splenocytes of ZARNAsome-treated mices were prepared.Subsequently, the amount of the surface expression of the maturationmarkers CD40 and CD86 on dendritic cells and macrophages was measuredvia flow cytometry. FIG. 7 shows that an increase of the signal of CD40and CD86 compared to the negative control (no treatment) was onlydetected in dendritic cells and in the macrophage cell population in thepresence of Luciferase (Luc) or influenza hemagglutinin A (InfHA) RNAdecorated zoledronic acid (ZA) encapsulating liposomes (ZARNAsomeLuc-RNA or ZARNAsome infHA-RNA) and Luciferase (Luc) or influenzahemagglutinin A (InfHA) RNA decorated buffer vehicle encapsulatingliposomes (EL+Luc-RNA or EL+InfHA-RNA). In contrast thereto, an increaseof the signal of CD40 and CD86 compared to the negative control (notreatment) was not detected in dendritic cells and in the macrophagecell population in the presence of undecorated zoledronic acid (ZA)encapsulating liposomes (ZA-L), buffer vehicle encapsulating liposomes(EL) or free RNAs (free Luc-RNA or free InfHA-RNA). These resultsindicate that treatment with RNA-decorated liposomes induces maturationof dendritic cells and macrophages in vivo.

As luciferase encoding RNA (Luc-RNA) as well as influenza hemagglutininA encoding RNA (InfHA-RNA) induced maturation of dendritic cells andmacrophages, the induction of maturation appears to be independent fromthe encoded protein. Therefore, any RNA can be used for decorating theparticles of the present invention in order to induce maturation ofsplenic dendritic cells and macrophages. This provides a useful methodin order to generally induce maturation of splenic dendritic cells andmacrophages as well as to introduce an antigen into splenic dendriticcells and macrophages which is specifically useful for vaccination orother immunotherapeutic approaches.

3.9 Application of Carboxyfluorescin (CF)—Filled Liposomes Decoratedwith Luc-RNA (CF-filled ZARNAsome) Leads to Transfection of Splenic CellPopulations Where Dendritic Cells (DCs) and Macrophages (mΦ) are theMain Target

Next, it was analyzed, whether the RNA decorated liposomes were taken upby splenic dendritic cells, macrophages, B cells or T cells. To monitorthe uptake of RNA decorated liposomes, liposomes filled with the dyecarboxyfluorescin (CF) and decorated with Luciferase (Luc) RNA wereinjected into mice. One hour after injection, splenocytes were preparedand analyzed using FACS analysis. FIG. 8 shows that injection ofcarboxyfluorescin-filled liposomes decorated with luciferase RNA(CF-filled ZARNAsome) in mice resulted in an uptake of said liposomes insplenic dendritic cells, macrophages, B cells and T cells, wherebydendritic cells and macrophages were the main targets. This indicatesthat the RNA decorated lipid particles of the present inventionpreferably target antigen presenting cells such as dendritic cells andmacrophages. Thus, the RNA decorated lipid particles of the presentinvention can be used for introducing RNA and therapeutic effectiveagents into dendritic cells and macrophages. In addition, dendriticcells and macrophages can be used for antigen presentation as well asfor immune response induction/modulation.

3.10 Zoledronic Acid Leads to Accumulation of Isopentenylpyrophosphate(IPP) in the Spleen.

After having shown that RNA provided by the ZARNAsomes is functionallyactive, the function of zoledronic acid encapsulated in the ZARNAsomewas tested in vivo.

Zoledronic acid has been shown to induce accumulation ofisopentenylpyrophosphate (IPP) in various cell lines in vitro and tumortissue in vivo and could be directly related to improved clinicaloutcome of cancer of different origin. (Mitrofan, L. M., Pelkonen, J.,Mönkkönen, J., (2009), Bone, 45, 1153-60). Thus, IPP accumulation in thespleen was investigated after injection of the liposome formulationsusing mass spectrometry. FIG. 9 shows that the treatment withundecorated zoledronic acid (ZA) encapsulating liposomes (ZA-L) andluciferase (Luc) RNA decorated zoledronic acid (ZA) encapsulatingliposomes (ZARNAsomes Luc-RNA) resulted in a significant increase of theIPP concentration in the spleen compared to the negative control (notreatment), free luciferase (Luc) RNA (free RNA), buffer vehicleencapsulating liposomes (EL) and buffer vehicle encapsulating liposomesdecorated with luciferase (Luc) encoding RNA (EL+Luc-RNA). Thisindicates that zoledronic acid delivered by ZARNAsomes is stillfunctional after delivery in vivo. Thus, the in vivo data confirmed theresults of the in vitro data, indicating that RNA decorated lipidparticles such as ZARNAsomes can be used for drug delivery.

In summary, it is shown by means of ZARNAsomes that RNA decorated lipidparticles are useful for introduction of protein such as antigenencoding RNA as well as for drug delivery in order to induce/modulatethe immune response in an individual.

ABBREVIATIONS

-   ZA=Zoledronic acid-   ZA-L=zoledronic acid encapsulating liposome-   ZARNAsome=RNA decorated zoledronic acid encapsulating liposome-   ZARNAsome Luc RNA=Luc RNA decorated zoledronic acid encapsulating    liposome-   ZARNAsome infHA RNA=infHA RNA decorated zoledronic acid    encapsulating liposome-   EL=buffer vehicle encapsulating liposome-   EL+RNA=RNA decorated buffer vehicle encapsulating liposome-   EL+Luc RNA=Luc RNA decorated buffer vehicle encapsulating liposome-   EL+infHA RNA=infHA RNA decorated buffer vehicle encapsulating    liposome-   Luc=Luciferase-   infHA=influenza hemagglutinin A-   CF=carboxyfluorescein-   IPP=Isopentenylpyrophosphate-   DOTMA=1,2-di-O-octadecenyl-3-trimethylammonium propane-   CHOL=Cholesterol-   POPC=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-   PI=polydispersity index-   Z_(ave)=Z-average-   i.v.=intra venous-   DC=dentritic cell-   iDC=immature dendritic cell-   PBMCs=peripheral blood mononuclear cells-   PBLs=peripheral blood lymphocytes-   MΦ=macrophage-   MFI=mean fluorescence intensity-   Cntr=control

1. A particle comprising: (i) a vesicular core, (ii) at least onetherapeutically effective compound encapsulated within the vesicularcore, and (iii) RNA forming a hydrophilic shell on at least a portion ofthe vesicular core.
 2. The particle of claim 1, wherein the RNA ispharmaceutically active or encodes at least one pharmaceutically activepeptide or protein.
 3. The particle of claim 1 or 2, wherein the RNAencodes at least one antigen.
 4. The particle of claim 3, wherein theantigen is a disease-associated antigen or elicits an immune responseagainst a disease-associated antigen or cells expressing adisease-associated antigen.
 5. The particle of any one of claims 1 to 4,wherein the RNA is exposed to surrounding medium.
 6. The particle of anyone of claims 1 to 5, wherein the RNA covers the entire surface of thevesicular core or a portion thereof.
 7. The particle of any one ofclaims 1 to 6, wherein the therapeutically effective compound is awater-soluble compound.
 8. The particle of any one of claims 1 to 7,wherein the therapeutically effective compound is a small moleculecompound.
 9. The particle of any one of claims 1 to 8, wherein thetherapeutically effective compound is useful in immunotherapy.
 10. Theparticle of any one of claims 1 to 9, wherein the therapeuticallyeffective compound is an agent stimulating γδ T cells, preferably Vγ9Vδ2T cells.
 11. The particle of claim 10, wherein the agent stimulating γδT cells is a bisphosphonate, preferably a nitrogen-containingbisphosphonate (aminobisphosphonate).
 12. The particle of claim 10 or11, wherein the agent stimulating γδ T cells is selected from the groupconsisting of zoledronic acid, clodronic acid, ibandronic acid,pamidronic acid, risedronic acid, minodronic acid, olpadronic acid,alendronic acid, incadronic acid and salts thereof.
 13. The particle ofany one of claims 1 to 12, wherein the vesicular core is positivelycharged.
 14. The particle of any one of claims 1 to 13, wherein thevesicular core is a polymer vesicular core, a protein vesicular core ora lipid vesicular core, preferably a lipid vesicular core.
 15. Theparticle of claim 14, wherein the lipid vesicular core comprises a lipidbilayer.
 16. The particle of claim 14 or 15, wherein the lipid vesicularcore comprises a liposome.
 17. The particle of any one of claims 14 to16, wherein the lipid vesicular core comprises at least one cationiclipid.
 18. The particle of claim 17, wherein the positive charges arecontributed by the at least one cationic lipid and the negative chargesare contributed by the RNA.
 19. The particle of any one of claims 14 to18, wherein the lipid vesicular core comprises at least one helperlipid.
 20. The particle of claim 19, wherein the helper lipid is aneutral lipid or negatively charged lipid.
 21. The particle of any oneof claims 17 to 20, wherein the at least one cationic lipid comprises1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or1,2-dioleoyl-3-trimethylammonium propane (DOTAP).
 22. The particle ofany one of claims 19 to 21, wherein the at least one helper lipidcomprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin(POPC) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 23. Theparticle of any one of claims 1 to 22, wherein the particle has anaverage diameter in the range of from about 50 nm to about 1000 nm. 24.The particle of claim 23, wherein the particle has an average diameter(i) in the range of from about 50 nm to about 400 nm, preferably fromabout 50 nm to about 200 nm, or (ii) in the range of from about 200 nmto about 1000 nm, preferably from about 200 nm to about 800 nm, morepreferably from about 300 nm to about 600 nm.
 25. The particle of anyone of claims 14 to 24, wherein the lipid vesicular core having thetherapeutically effective compound encapsulated therein is obtainable byreverse phase evaporation technique or ethanol injection technique. 26.The particle of any one of claims 14 to 25, wherein the particle isobtainable by addition of the RNA to a lipid vesicular core having thetherapeutically effective compound encapsulated therein.
 27. Theparticle of any one of claims 14 to 26, wherein the particle isobtainable by a process comprising a step of extruding and/or a step oflyophilizing the particle.
 28. A pharmaceutical composition comprisingparticles as set forth in any one of claims 1 to
 27. 29. Thepharmaceutical composition of claim 28, wherein, after systemicadministration of the particles, at least a portion of the RNA and atleast a portion of the therapeutically effective compound are deliveredto a target cell, preferably to the same target cell.
 30. Thepharmaceutical composition of claim 29, wherein the target cell is aspleen cell, preferably an antigen presenting cell, more preferably aprofessional antigen presenting cell, more preferably a dendritic cell.31. The pharmaceutical composition of any one of claims 28 to 30,wherein, after systemic administration of the particles, RNAaccumulation and/or RNA expression in the spleen occurs.
 32. Thepharmaceutical composition of any one of claims 28 to 31, wherein, aftersystemic administration of the particles, no or essentially no RNAaccumulation and/or RNA expression in the lung and/or liver occurs. 33.The pharmaceutical composition of any one of claims 28 to 32, wherein,after systemic administration of the particles, RNA accumulation and/orRNA expression in the spleen is at least 5-fold the amount of RNAaccumulation and/or RNA expression in the lung.
 34. The pharmaceuticalcomposition of any one of claims 28 to 33, wherein, after systemicadministration of the particles, RNA accumulation and/or RNA expressionin antigen presenting cells, preferably professional antigen presentingcells in the spleen occurs.
 35. The pharmaceutical composition of claim34, wherein the antigen presenting cells are dendritic cells and/ormacrophages.
 36. The pharmaceutical composition of any one of claims 29to 35, wherein systemic administration is by parenteral administration,preferably by intravenous administration, subcutaneous administration,intradermal administration or intraarterial administration.
 37. Thepharmaceutical composition of any one of claims 28 to 36, wherein thecomposition further comprises one or more pharmaceutically acceptablecarriers, diluents and/or excipients.
 38. The pharmaceutical compositionof any one of claims 28 to 37, wherein the composition further comprisesat least one adjuvant.
 39. The pharmaceutical composition of any one ofclaims 28 to 38, wherein the composition is formulated for systemicadministration.
 40. The pharmaceutical composition of any one of claims28 to 39 for inducing or enhancing an immune response, preferably animmune response against cancer.
 41. The pharmaceutical composition ofany one of claims 28 to 40 for use in a prophylactic and/or therapeutictreatment of a disease involving an antigen, preferably a cancerdisease.
 42. A method for delivering an antigen to antigen presentingcells, preferably professional antigen presenting cells, in the spleen,or expressing an antigen in antigen presenting cells, preferablyprofessional antigen presenting cells, in the spleen comprisingadministering to a subject a pharmaceutical composition of any one ofclaims 28 to
 39. 43. The method of claim 42, wherein the antigenpresenting cells are dendritic cells and/or macrophages.
 44. A methodfor inducing or enhancing an immune response, preferably an immuneresponse against cancer, in a subject comprising administering to thesubject a pharmaceutical composition of any one of claims 28 to
 39. 45.A method for stimulating, priming and/or expanding T cells in a subjectcomprising administering to the subject a pharmaceutical composition ofany one of claims 28 to
 39. 46. A method of treating or preventing adisease involving an antigen, preferably a cancer disease, in a subjectcomprising administering to the subject a pharmaceutical composition ofany one of claims 28 to
 39. 47. A method of producing a particle of anyone of claims 1 to 27 comprising the following steps of: (i) providing avesicular core having at least one therapeutically effective compoundencapsulated therein, and (ii) adding RNA to the vesicular core, whereinthe RNA forms a hydrophilic shell on at least a portion of the vesicularcore, thereby forming the particle.
 48. The method of claim 47, whereinthe lipid vesicular core to which the RNA is added comprises a liposomecomprising at least one cationic lipid.
 49. The method of claim 48,wherein the amount of RNA and the amount of cationic lipids in theliposome is selected such that the net charge formed by the positivecharges derived from the cationic lipids and the negative chargesderived from the RNA is negative, positive, or zero.
 50. The method ofclaim 49, wherein the number of positive charges derived from thecationic lipids divided by the number of negative charges derived fromthe RNA is between 0.025 and 4.